Ionizable cationic lipids

ABSTRACT

Disclosed herein are novel compounds, pharmaceutical compositions comprising such compounds and related methods of their use. The compounds described herein are useful, e.g., as liposomal delivery vehicles to facilitate the delivery of encapsulated polynucleotides to target cells and subsequent transfection of said target cells, and in certain embodiments are characterized as having one or more properties that afford such compounds advantages relative to other similarly classified lipids.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/368,280, filed Dec. 2, 2016, which is a continuation of U.S.application Ser. No. 14/389,023, filed Sep. 29, 2014, and which issuedas U.S. Pat. No. 9,546,128 on Jan. 17, 2017, which is the U.S. NationalStage of International Application No. PCT/US2013/034602, filed Mar. 29,2013, which claims the benefit of U.S. Provisional Application No.61/617,468, filed on Mar. 29, 2012, the disclosures of which areincorporated herein by reference in their entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Aug. 9, 2018, isnamed MRT-1060US3_ST25.txt and is 4,212 bytes in size.

BACKGROUND

Liposomal delivery of nucleic acids has been employed as a means ofeffectuating the site-specific delivery of encapsulated plasmid DNA,antisense oligonucleotides, short interfering RNA and microRNA-basedtherapies, however the efficient delivery of nucleic acids to targetedcells and tissues, as well as the subsequent transfection of suchtargeted cells and tissues remains a technical challenge. Despite theavailability of multiple liposomal-based systems and vehicles tofacilitate the delivery of therapeutic agents to target cells andtissues, many problems still exist both in in vivo and in vitroapplications. For example, a significant drawback of liposomal deliverysystems relates to the construction of liposomes that have sufficientcell culture or in vivo stability to reach desired target cells and/orintracellular compartments, and the ability of such liposomal deliverysystems to efficiently release their encapsulated materials to suchtarget cells.

Furthermore, many of the cationic lipids that are employed to constructsuch liposomal-based vehicles are generally toxic to the targeted cells.In particular, the amount of such cationic lipid that is necessary todeliver a therapeutically effective amount of the encapsulated agent maybe toxic to the targeted cells. Accordingly, the toxicity associatedwith cationic lipid represents a significant obstacle to their generaluse as non-viral vectors, particularly in the quantities necessary tosuccessfully deliver therapeutically effective amounts of theencapsulated materials to target cells.

Despite the foregoing limitations, and as a results of their ability toprotect and facilitate the delivery of encapsulated materials to one ormore target cells, liposomal-based vehicles are considered an attractivecarrier for therapeutic agents and remain subject to continueddevelopment efforts. While liposomal-based vehicles that comprise acationic lipid component have shown promising results with regards toencapsulation, stability and site localization, there remains a greatneed for improvement of liposomal-based delivery systems. In particular,there remains a need for improved cationic and ionizable lipids thatdemonstrate improved pharmacokinetic properties and which are capable ofdelivering macromolecules, such as nucleic acids to a wide variety celltypes and tissues with enhanced efficiency. Importantly, there alsoremains a particular need for novel cationic ionizable lipids that arecharacterized as having reduced toxicity and are capable of efficientlydelivering encapsulated nucleic acids and polynucleotides to targetedcells, tissues and organs.

SUMMARY

Described herein are novel cationic and ionizable lipid compounds,pharmaceutical compositions comprising such compounds and relatedmethods of their use. In certain embodiments, the compounds describedherein are useful as liposomal compositions or as components ofliposomal compositions to facilitate the delivery to, and subsequenttransfection of one or more target cells. In certain embodiments, thelipid compositions disclosed herein are cationic and/or ionizablelipids. For example, the lipid compounds disclosed herein may comprise abasic ionizable functional group such as an amine. In some embodiments,the compounds described herein have been designed based on one or moredesired characteristics or properties, for example to enhancetransfection efficiency or to promote specific biological outcomes.

In certain embodiments disclosed herein, the lipid compounds generallycomprise a polar, hydrophilic head-group and a non-polar, hydrophobictail-group. For example, the lipid compounds disclosed herein maygenerally comprise one or more cationic and/or ionizable functionalhead-groups, such as an amine functional group having one or more alkylor aryl substituents. In certain embodiments the lipid compoundsdisclosed herein may comprise a cationic ionizable amino functionalhead-group to which is bound (e.g., covalently bound) two alkylfunctional groups, substituents or moieties (e.g., an R₁ group and a R₂group, wherein both R₁ and R₂ are independently selected from the groupconsisting of C₁-C₁₀ alkyls).

In some embodiments the hydrophilic head-group (e.g., an alkyl aminogroup) is bound (e.g., covalently bound) to a hydrophobic (lipophilic)tail-group. For example, the lipophilic tail-group (e.g., one or more ofan L₁ group and an L₂ group) of the compounds disclosed herein maycomprise one or more non-polar groups such as cholesterol or anoptionally substituted, variably unsaturated alkyl (e.g., an optionallysubstituted octadeca-9,12-diene or octadec-6,9-diene).

In certain embodiments, the present invention relates to compoundshaving the structure of formula (I):

wherein R₁ and R₂ are each independently selected from the groupconsisting of hydrogen, an optionally substituted, variably saturated orunsaturated C₁-C₂₀ alkyl and an optionally substituted, variablysaturated or unsaturated C₆-C₂₀ acyl; wherein L₁ and L₂ are eachindependently selected from the group consisting of hydrogen, anoptionally substituted C₁-C₃₀ alkyl, an optionally substituted variablyunsaturated C₁-C₃₀ alkenyl, and an optionally substituted C₁-C₃₀alkynyl; wherein m and o are each independently selected from the groupconsisting of zero and any positive integer (e.g., where m is three);and wherein n is zero or any positive integer (e.g., where n is one).

In certain embodiments, the compound has the structure of formula (I),wherein R₁ and R₂ are each methyl. In such embodiment, the polarcationic head-group of the compound comprises an ionizable dimethylamino group.

In some embodiments, the compound has the structure of formula (I),wherein L₁ and L₂ are each an optionally substituted, polyunsaturatedC₆-C₂₀ alkenyl. For example, contemplated are compounds wherein L₁ andL₂ are each an optionally substituted polyunsaturated C₁₈ alkenyl. Inother embodiments, L₁ and L₂ are each an unsubstituted, polyunsaturatedC₁₈ alkenyl. In yet other embodiments, L₁ and L₂ are each an optionallysubstituted octadeca-9,12-diene (or octadec-6,9-diene). In still otherembodiments, L₁ is hydrogen and L₂ is cholesterol.

In certain embodiments disclosed herein, the present inventions relateto a compound having the structure of formula (I), wherein o is zero.Alternatively, in other embodiments, o is a positive integer (e.g., one,two, three, four, five, six, seven, eight, nine, ten, eleven, twelve,thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen,twenty, or more).

In certain embodiments disclosed herein, the present inventions relateto a compound having the structure of formula (I), wherein m is apositive integer (e.g., one, two, three, four, five, six, seven, eight,nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen,seventeen, eighteen, nineteen, twenty, or more). In some particularembodiments, the present inventions relate to a compound having thestructure of formula (I), wherein m is four. In some particularembodiments, the present inventions relate to a compound having thestructure of formula (I), wherein m is three.

Also disclosed herein are compounds having the structure of formula (I),wherein n is a positive integer (e.g., one, two, three, four, five, six,seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen,sixteen, seventeen, eighteen, nineteen, twenty, or more). In otherparticular embodiments, the present inventions relate to a compoundhaving the structure of formula (I), wherein n is zero.

In some particular embodiments, the present invention relates to acompound having the structure of formula (I), wherein R₁ and R₂ are eachmethyl; wherein L₁ and L₂ are each octadeca-9,12-diene (oroctadec-6,9-diene); wherein m is four; wherein n is zero; and wherein ois one. For example, in certain embodiments, the present inventionrelates to the compound(15Z,18Z)—N,N-dimethyl-6-(9Z,12Z)-octadeca-9,12-dien-1-yl)tetracosa-15,18-dien-1-amine.In certain embodiments, the present invention relates to a compoundhaving the structure of formula (II), (referred to herein as “HGT5000”).

In some particular embodiments, the present invention relates to acompound having the structure of formula (I), wherein R₁ and R₂ are eachmethyl; wherein L₁ and L₂ are each octadeca-9,12-diene (oroctadec-6,9-diene); wherein m is 3; wherein n is one; and wherein o iszero. For example, in certain embodiments, the present invention relatesto the compound(15Z,18Z)—N,N-dimethyl-6-((9Z,12Z)-octadeca-9,12-dien-1-yl)tetracosa-4,15,18-trien-1-amine.In certain embodiments, the present invention relates to a compoundhaving the structure of formula (III), (referred to herein as“HGT5001”).

It should be understood that in those embodiments disclosed herein wheren is one, such compounds may be a cis isomer, a trans isomer oralternatively a racemic mixture thereof. For example, in certainembodiments where n is one, n is a cis isomer, as represented by acompound having the structure of formula (IV):

Alternatively, in other embodiments where n is one, n is a trans isomer,as represented by a compound having the structure of formula (V):

Also disclosed are compounds having the structure of formula (VI):

wherein R₁ and R₂ are each independently selected from the groupconsisting of hydrogen, an optionally substituted, variably saturated orunsaturated C₁-C₂₀ alkyl and an optionally substituted, variablysaturated or unsaturated C₆-C₂₀ acyl; wherein L₁ and L₂ are eachindependently selected from the group consisting of hydrogen, anoptionally substituted C₁-C₃₀ alkyl, an optionally substituted variablyunsaturated C₁-C₃₀ alkenyl, and an optionally substituted C₁-C₃₀alkynyl; and wherein m, n and o are each independently selected from thegroup consisting of zero and any positive integer.

In some particular embodiments, the present inventions are directed to acompound having the structure of formula (VI), wherein R₁ and R₂ areeach methyl. In other embodiments, the present inventions are directedto a compound having the structure of formula (VI), wherein R₁ and R₂are each independently selected from the group consisting of hydrogenand methyl.

Also contemplated are compounds having the structure of formula (VI),wherein L₁ and L₂ are each an optionally substituted, polyunsaturatedC₆-C₂₀ alkenyl (e.g., where L₁ and L₂ are each an optionally substitutedpolyunsaturated C₁₈ alkenyl or where L₁ and L₂ are each anunsubstituted, polyunsaturated C₁₈ alkenyl). In certain embodiments,disclosed herein, L₁ and L₂ are each an optionally substitutedoctadeca-9,12-diene (or octadec-6,9-diene). In other embodiments L₁ ishydrogen and L₂ is cholesterol.

In certain embodiments disclosed herein, the present inventions relateto a compound having the structure of formula (VI), wherein m is apositive integer (e.g., one, two, three, four, five, six, seven, eight,nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen,seventeen, eighteen, nineteen, twenty, or more). In some particularembodiments, the present inventions relate to a compound having thestructure of formula (VI), wherein m is four. In certain embodiments,the present inventions relate to a compound having the structure offormula (VI), wherein m is at least five (e.g., where m is five, six,seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen,sixteen, seventeen, eighteen, nineteen, twenty or more).

Also disclosed herein are compounds having the structure of formula(VI), wherein n is a positive integer (e.g., one, two, three, four,five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen,fifteen, sixteen, seventeen, eighteen, nineteen, twenty, or more). Inother particular embodiments, the present inventions relate to acompound having the structure of formula (VI), wherein n is zero.

In certain embodiments disclosed herein, the present inventions aredirected to compounds having the structure of formula (VI), wherein o isa positive integer (e.g., one, two, three, four, five, six, seven,eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen,seventeen, eighteen, nineteen, twenty, or more). In certain embodiments,the present inventions relate to a compound having the structure offormula (VI), wherein o is at least five (e.g., where o is five, six,seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen,sixteen, seventeen, eighteen, nineteen, twenty or more). Alternatively,in other particular embodiments, the present inventions relate tocompounds having the structure of formula (VI), wherein o is zero.

Also contemplated are compounds having the structure of formula (VI),wherein R₁ and R₂ are each methyl; wherein L₁ and L₂ are eachoctadeca-9,12-diene (or octadec-6,9-diene); wherein m is 4; and whereinboth n and o are zero. For example, in certain embodiments, the presentinvention relates to the compound(15Z,18Z)—N,N-dimethyl-6-((9Z,12Z)-octadeca-9,12-dien-1-yl)tetracosa-5,15,18-trien-1-amine.In certain embodiments, the present invention relates to the compoundhaving the structure of formula (VII), (referred to herein as“HGT5002”):

Also disclosed herein are compounds having the structure of formula(VIII):

wherein R₁ and R₂ are each independently selected from the groupconsisting of an optionally substituted, variably saturated orunsaturated C₁-C₂₀ alkyl or alkenyl and an optionally substituted,variably saturated or unsaturated C₆-C₂₀ acyl; wherein L₁ and L₂ areeach independently selected from the group consisting of an optionallysubstituted C₁-C₃₀ alkyl, an optionally substituted variably unsaturatedC₁-C₃₀ alkenyl, and an optionally substituted C₁-C₃₀ alkynyl; andwherein x is selected from the group consisting of a C₁-C₂₀ alkyl and avariably unsaturated C₁-C₂₀ alkenyl.

In certain embodiments, the disclosed compounds have the structure offormula (VIII), wherein R₁ and R₂ are each methyl. In other embodiments,the disclosed compounds have the structure of formula (VIII), wherein R₁and R₂ are independently selected from the group consisting of hydrogenand a C₁-C₆ alkyl.

In other embodiments, the present invention relates to compounds havingthe structure of formula (VIII), wherein L₁ and L₂ are each anunsubstituted, polyunsaturated C₁₈ alkenyl. For example, in certainembodiments, L₁ and L₂ are each an optionally substitutedoctadeca-9,12-diene (e.g., L₁ and L₂ are each an unsubstitutedoctadeca-9,12-diene or octadec-6,9-diene). In certain other embodiments,L₁ is hydrogen and L₂ is cholesterol.

In certain embodiments, the disclosed compounds have the structure offormula (VIII), wherein x is a C₆ alkenyl. In other embodiments, thedisclosed compounds have the structure of formula (VIII), wherein x ishexane. In yet other embodiments, the disclosed compounds have thestructure of formula (VIII), wherein x is hex-1-ene. In still otherembodiments, the disclosed compounds have the structure of formula(VIII), wherein x is hex-2-ene. In certain embodiments, x is not hexane.In other embodiments, the disclosed compounds have the structure offormula (VIII), wherein x is a C₆₋₁₀ alkenyl or a C₆₋₁₀ alkyl.

In one particular embodiment, the present invention relates to acompound having the structure of formula (VIII), wherein R₁ and R₂ areeach methyl; wherein L₁ and L₂ are each octadeca-9,12-diene (oroctadec-6,9-diene); and wherein x is hexane. In another particularembodiment, the present invention relates to a compound having thestructure of formula (VIII), wherein R₁ and R₂ are each methyl; whereinL₁ and L₂ are each octadeca-9,12-diene (or octadec-6,9-diene); andwherein x is hex-1-ene. In still another particular embodiment, thepresent invention relates to a compound having the structure of formula(VIII), wherein R₁ and R₂ are each methyl; wherein L₁ and L₂ are eachoctadeca-9,12-diene (or octadec-6,9-diene); and wherein x is hex-2-ene.

It should be understood that in those embodiments described herein wherethe compounds have one or more asymmetric or chiral molecules (e.g., oneor more unsaturated carbon-carbon double bonds), both the cis (Z) andtrans (E) isomers are within the scope of this invention.

The compositions disclosed herein may be used to prepare one or morepharmaceutical compositions and/or liposomal vehicles (e.g., a lipidnanoparticle). In such embodiments, such pharmaceutical compositions orvehicles may further comprise one or more compounds selected from thegroup consisting of a cationic lipid, a PEG-modified lipid, anon-cationic lipid and a helper lipid. Accordingly, in certainembodiments, the compounds described herein (e.g., HGT5000, HGT5001,and/or HGT5002) are cationic or ionizable lipids that may be used as acomponent of a liposomal composition to facilitate or enhance thedelivery and release of encapsulated materials (e.g., one or moretherapeutic agents) to one or more target cells (e.g., by permeating orfusing with the lipid membranes of such target cells). Enrichingliposomal compositions with one or more of the compounds disclosedherein may be used as a means of improving (e.g., reducing) the toxicityor otherwise conferring one or more desired properties to such enrichedliposomal composition (e.g., improved delivery of the encapsulatedpolynucleotides to one or more target cells and/or reduced in vivotoxicity of a liposomal composition). Accordingly, also contemplated arepharmaceutical compositions, and in particular liposomal compositions,that comprise one or more of the compounds disclosed herein. In certainembodiments, such pharmaceutical and liposomal compositions comprise oneor more of a PEG-modified lipid, a non-cationic lipid and a helperlipid. For example, contemplated are pharmaceutical and liposomalcompositions (e.g., lipid nanoparticles) that comprise one or more ofthe compounds disclosed herein (e.g., HGT5000, HGT5001, and/or HGT5002)and one or more helper lipids, non-cationic lipids and PEG-modifiedlipids components. Also contemplated are pharmaceutical and liposomalcompositions that comprise one or more of the compounds disclosed hereinand that further comprise one or more additional cationic lipids.Similarly, also contemplated are liposomal compositions andpharmaceutical compositions (e.g., a lipid nanoparticle) that compriseone or more of the HGT5000, HGT5001 and/or HGT5002 compounds and one ormore of C12-200, DLinDMA, CHOL, DOPE, DMG-PEG-2000, ICE, DSPC, DODAP,DOTAP and C8-PEG-2000. In certain embodiments, such pharmaceuticalcompositions and liposomal compositions are loaded with or otherwiseencapsulate materials, such as for example, one or morebiologically-active polynucleotides.

In certain embodiments one or more of the pharmaceutical and liposomalcompositions described herein (e.g., lipid nanoparticles) comprise oneor more of the compounds disclosed herein and one or more additionallipids. For example, lipid nanoparticles that comprise or are otherwiseenriched with one or more of the compounds disclosed herein may furthercomprise one or more of DOTAP (1,2-dioleyl-3-trimethylammonium propane),DODAP (1,2-dioleyl-3-dimethylammonium propane), DOTMA(1,2-di-O-octadecenyl-3-trimethylammonium propane), DLinDMA,DLin-KC2-DMA, C12-200 and ICE. In one embodiment the pharmaceuticalcomposition comprises a lipid nanoparticle that comprises HGT5000, DOPE,cholesterol and/or DMG-PEG2000. In another embodiment the pharmaceuticalcomposition comprises a lipid nanoparticle that comprises HGT5001, DOPE,cholesterol and/or DMG-PEG2000. In yet another embodiment thepharmaceutical composition comprises a lipid nanoparticle that comprisesHGT5002, DOPE, cholesterol and/or DMG-PEG2000.

In certain embodiments one or more of the pharmaceutical compositionsdescribed herein may comprise one or more PEG-modified lipids. Forexample, lipid nanoparticles that comprise or are otherwise enrichedwith one or more of the compounds disclosed herein may further compriseone or more of PEG-modified lipids that comprise a poly(ethylene)glycolchain of up to 5 kDa in length covalently attached to a lipid comprisingone or more C₆-C₂₀ alkyls.

Similarly, the pharmaceutical compositions disclosed herein (e.g., lipidnanoparticles) may comprise or may otherwise be enriched with one ormore of the compounds disclosed herein and may further comprise one ormore of helper lipids that are selected from the group consisting ofDSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), DPPC(1,2-dipalmitoyl-sn-glycero-3-phosphocholine), DOPE(1,2-dioleyl-sn-glycero-3-phosphoethanolamine), DPPE(1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine), DMPE(1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine), DOPG(,2-dioleoyl-sn-glycero-3-phospho-(1′-rac-glycerol)), DOPE(1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), DSPE(1,2-distearoyl-sn-glycero-3-phosphoethanolamine), DLPE(1,2-dilauroyl-sn-glycero-3-phosphoethanolamine), DPPS(1,2-dipalmitoyl-sn-glycero-3-phospho-L-serine), ceramides,sphingomyelins and cholesterol.

In certain embodiments, the compounds and the pharmaceutical andliposomal compositions comprising such compounds (e.g., lipidnanoparticles) comprise one or more polynucleotides (e.g., encapsulatedDNA or RNA). In other embodiments, the one or more polynucleotidescomprise at least one locked nucleic acid (e.g., two, three, four, five,six, seven, eight, nine, ten, twelve, fifteen, sixteen, eighteen,twenty, or more locked nucleic acid residues or monomers). Where the oneor more encapsulated polynucleotides comprise RNA, such RNA may include,for example, mRNA, siRNA, snoRNA, microRNA, and combinations thereof.

In certain embodiments, the polynucleotides encapsulated in thepharmaceutical and liposomal compositions hereof comprise mRNA encoding,for example, a functional polypeptide, protein or enzyme, and upon beingexpressed (i.e., translated) by one or more target cells a functionalexpression product (e.g., a polypeptide, protein or enzyme) is produced,and in some instances secreted by the target cell into the peripheralcirculation (e.g., plasma) of a subject. In certain embodiments, the oneor more of the polynucleotides that comprise (or are otherwise loaded orencapsulated into) the compounds and pharmaceutical and liposomalcompositions described herein encode a nucleic acid (e.g., apolypeptide) which is aberrantly expressed by the subject. In certainembodiments, the one or more of the encapsulated polynucleotides thatcomprise such compounds and liposomal or pharmaceutical compositions(e.g., a lipid nanoparticle) encode a functional protein or enzyme. Forexample, the polynucleotide (e.g., mRNA) may encode a protein or enzymeselected from the group consisting of erythropoietin, human growthhormone, cystic fibrosis transmembrane conductance regulator (CFTR),alpha-glucosidase, arylsulfatase A, alpha-galactosidase A,alpha-L-iduronidase, iduronate-2-sulfatase, iduronate sulfatase,N-acetylglucosamine-1-phosphate transferase, N-acetylglucosaminidase,alpha-glucosaminide acetyltransferase, N-acetylglucosamine 6-sulfatase,N-acetylgalactosamine-4-sulfatase, beta-glucosidase, galactose-6-sulfatesulfatase, beta-galactosidase, beta-glucuronidase, glucocerebrosidase,heparan sulfamidase, heparin-N-sulfatase, lysosomal acid lipase,hyaluronidase, galactocerebrosidase, ornithine transcarbamylase (OTC),carbamoyl-phosphate synthetase 1 (CPS1), argininosuccinate synthetase(ASS1), argininosuccinate lyase (ASL), and arginase 1 (ARG1).

In certain embodiments the encapsulated polynucleotide encodes anenzyme, such enzyme may be a urea cycle enzyme (e.g., ornithinetranscarbamylase (OTC), carbamoyl-phosphate synthetase 1 (CPS1),argininosuccinate synthetase (ASS1), argininosuccinate lyase (ASL) orarginase 1 (ARG1)). In certain embodiments the one or more of theencapsulated polynucleotides comprises mRNA encoding an enzymeassociated with a lysosomal storage disorder (e.g., the encapsulatedpolynucleotide is mRNA encoding one or more of the enzymesα-galactosidase A, iduronate-2-sulfatase, iduronate sulfatase,N-acetylglucosamine-1-phosphate transferase, beta-glucosidase andgalactocerebrosidase).

Also contemplated herein are pharmaceutical and liposomal compositions(e.g., lipid nanoparticles) that comprise one or more of the compoundsdisclosed herein and one or more polynucleotides (e.g., antisenseoligonucleotides), and in particular polynucleotides that comprises oneor more chemical modifications. Contemplated polynucleotidemodifications may include, for example, sugar modifications orsubstitutions (e.g., one or more of a 2′-O-alkyl modification, a lockedpolynucleotide (LNA) or a peptide polynucleotide (PNA)). In embodimentswhere the sugar modification is a 2′-O-alkyl modification, suchmodification may include, but are not limited to a 2′-deoxy-2′-fluoromodification, a 2′-O-methyl modification, a 2′-O-methoxyethylmodification and a 2′-deoxy modification. In certain embodiments wherethe modification is a nucleobase modification, such modification may beselected from the group consisting of a 5-methyl cytidine,pseudouridine, 2-thio uridine, 5-methylcytosine, isocytosine,pseudoisocytosine, 5-bromouracil, 5-propynyluracil, 6-aminopurine,2-aminopurine, inosine, diaminopurine and 2-chloro-6-aminopurinecytosine, and combinations thereof.

In those embodiments where the polynucleotide is mRNA, such chemicalmodifications preferably render the mRNA more stable (e.g., moreresistant to nuclease degradation) and may comprise, for example an endblocking modification of a 5′ or 3′untranslated region of the mRNA. Incertain embodiments, the chemical modification comprises the inclusionof a partial sequence of a CMV immediate-early 1 (IE1) gene to the 5′untranslated region of the mRNA. In other embodiments the chemicalmodification comprises the inclusion of a poly A tail to the 3′untranslated region of the mRNA. Also contemplated are chemicalmodifications that comprise the inclusion of a Cap1 structure to the 5′untranslated region of the mRNA. In still other embodiments, thechemical modification comprises the inclusion of a sequence encodinghuman growth hormone (hGH) to the 3′ untranslated region of the mRNA.

The compounds and pharmaceutical compositions described herein may beformulated to specifically target and/or transfect one or more targetcells, tissues and organs. In certain embodiments, such compounds andpharmaceutical compositions facilitate the transfection of such targetcells by one or more mechanisms (e.g., fusogenic-based release and/orproton-sponge mediated disruption of the lipid-bilayer membrane of thetarget cells). Contemplated target cells include, for example, one ormore cells selected from the group consisting of hepatocytes,hematopoietic cells, epithelial cells, endothelial cells, lung cells,bone cells, stem cells, mesenchymal cells, neural cells, cardiac cells,adipocytes, vascular smooth muscle cells, cardiomyocytes, skeletalmuscle cells, beta cells, pituitary cells, synovial lining cells,ovarian cells, testicular cells, fibroblasts, B cells, T cells,reticulocytes, leukocytes, granulocytes and tumor cells.

Also disclosed are methods of treating disease (e.g., a diseaseassociated with the aberrant expression of a gene or nucleic acid) in asubject, wherein the method comprises administering one or more of thecompounds and/or pharmaceutical compositions described herein to thesubject. Also contemplated are methods of transfecting one or moretarget cells with one or more polynucleotides, wherein the methodcomprises contacting the one or more target cells with the compounds orpharmaceutical composition described herein such that the one or moretarget cells are transfected with the one or more polynucleotidesencapsulated therein.

The above discussed and many other features and attendant advantages ofthe present invention will become better understood by reference to thefollowing detailed description of the invention when taken inconjunction with the accompanying examples. The various embodimentsdescribed herein are complimentary and can be combined or used togetherin a manner understood by the skilled person in view of the teachingscontained herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. illustrates the concentration of human alpha-galactosidase (GLA)protein detected in the serum of wild type (WT) mice administered twosingle 90 μg, 60 μg, 30 μg, 20 μg or 10 μg intravenous doses of GLA mRNAencapsulated in an HGT5000-based lipid nanoparticle over a one weekperiod, at day one and again at day five. The serum concentrations ofGLA protein were determined at six hours, twenty-four hours, forty-eighthours and seventy-two hours following the administration of the secondintravenous dose. The mice were sacrificed seventy-two hours followingthe administration of the second intravenous dose on day eight. As shownin FIG. 1, following the intravenous injection of the second dose of GLAmRNA encapsulated in the HGT5000-based lipid nanoparticles, asubstantial level of human GLA protein could be detected in mouse serumwithin six hours and GLA protein was further detectable forty-eighthours post-administration.

FIG. 2. depicts the concentration of human alpha-galactosidase (GLA)protein detected in the liver, kidney and spleen of wild type (WT) miceadministered two single 90 μg, 60 μg, 30 μg, 20 μg or 10 μg doses of GLAmRNA encapsulated in an HGT5000-based lipid nanoparticle over a one weekperiod, at day one and again at day five. The mice were sacrificedseventy-two hours following the administration of the second intravenousdose on day eight and the concentration of GLA protein in the liver,kidneys and spleen of the wild type (WT) mice was determined. Asillustrated in FIG. 2, nanogram concentrations of human GLA protein weredetectable in the liver, kidney and spleen following administration ofthe GLA mRNA.

FIG. 3. illustrates the concentration of human alpha-galactosidase (GLA)protein detected in the serum of a murine model of Fabry disease over aseventy-two hour period following the intravenous administration of asingle 90 μg intravenous dose of GLA mRNA encapsulated in anHGT5000-based lipid nanoparticle. Supraphysiological concentrations ofGLA protein were detected in the serum of the Fabry mice twenty-fourhours following the administration of a single 90 μg dose of the GLAmRNA encapsulated in an HGT5000-based lipid nanoparticle.

FIG. 4. depicts the concentration of human alpha-galactosidase (GLA)protein detected in the liver, kidney, spleen and heart of a murinemodel of Fabry disease at twenty-four and seventy-two hours followingthe intravenous administration of a single dose of GLA encapsulated inan HGT5000-based lipid nanoparticle. GLA protein was detectable in theevaluated organs of the Fabry mouse at twenty-four and seventy-two hourspost-administration of the GLA mRNA, as shown in FIG. 4.

FIG. 5. illustrates the concentrations of human alpha-galactosidase(GLA) protein detected in wild type (WT) mouse serum over a twenty-fourhour period following the intravenous injection of a 30 μg dose of GLAmRNA encapsulated in an HGT5001-based lipid nanoparticle. As depicted inFIG. 5, within six hours of administration of the GLA mRNA, human GLAprotein was detected in serum at concentrations exceeding normalphysiological levels by 100-fold. Similarly, within twenty-four hoursfollowing administration of the GLA mRNA, human GLA protein was detectedin serum at concentrations exceeding normal physiological levels by30-fold.

FIG. 6. illustrates the concentrations of human alpha-galactosidase(GLA) protein detected in the liver, kidney and spleen of wild type (WT)mice over a twenty-four hour period following the intravenous injectionof GLA mRNA encapsulated in an HGT5001-based lipid nanoparticle. Asdepicted in FIG. 6, substantial levels of human GLA protein could bedetected in the liver, kidney and spleen of the WT mice twenty-fourhours following the intravenous administration of GLA mRNA encapsulatedin an HGT5001-based lipid nanoparticle.

FIG. 7. compares the serum concentrations of human erythropoietin (EPO)protein detected in Sprague-Dawley rats following the intravenousadministration of a single dose of EPO mRNA encapsulated in either anHGT5000- or an HGT5001-based lipid nanoparticle over a twenty-four hourperiod. As illustrated in FIG. 7, significant concentrations of EPOprotein were detected at six, twelve, eighteen and twenty-four hoursfollowing the intravenous administration of the EPO mRNA in both theHGT5000- and HGT5001-based lipid nanoparticles.

DETAILED DESCRIPTION

Disclosed herein are novel compounds that are useful, for example, asliposomal delivery vehicles or as components of liposomal deliveryvehicles. In certain embodiments, the compounds disclosed herein may beused as a liposomal composition or alternatively as component of aliposomal composition (e.g., as a lipid nanoparticle). Also disclosedare pharmaceutical compositions (e.g., lipid nanoparticles) and methodsof use relating to such pharmaceutical compositions. In certainembodiments, such compounds and compositions facilitate the delivery of,for example, encapsulated materials (e.g., polynucleotides) to one ormore target cells, tissues and organs.

The cationic and/or ionizable compounds disclosed herein generallycomprise both a polar (hydrophilic) head-group or moiety and a non-polar(hydrophobic or lipophilic) tail-group or moiety. In certainembodiments, such polar head-group and non-polar tail-group aregenerally bound (e.g., bound by one or more of hydrogen-bonds, van derWaals' forces, ionic interactions and covalent bonds) to each other(e.g., a head-group and a tail-group covalently bound to each other byan optionally substituted, variably unsaturated C₁-C₁₀ alkyl oralkenyl). In certain embodiments, the head-group or moiety ishydrophilic (e.g., a hydrophilic head-group comprising anoptionally-substituted alkyl amino). As used herein, the term“hydrophilic” is used to indicate in qualitative terms that a functionalgroup is water-preferring, and typically such groups are water-soluble.For example, disclosed herein are compounds that comprise a variablyunsaturated alkyl functional group bound to one or more hydrophilicgroups (e.g., a hydrophilic head-group), wherein such hydrophilic groupscomprise an amino group or an optionally-substituted alkyl amino group.

In certain embodiments, the selected hydrophilic functional group ormoiety may alter or otherwise impart properties to the compound or tothe liposomal composition of which such compound is a component (e.g.,by improving the transfection efficiencies of a lipid nanoparticle ofwhich the compound is a component). For example, the incorporation ofamino group as a hydrophilic head-group in the compounds disclosedherein may promote the fusogenicity of such compound (or of theliposomal composition of which such compound is a component) with thecell membrane of one or more target cells, thereby enhancing, forexample, the transfection efficiencies of such compound. Similarly, theincorporation of one or more alkyl amino groups or moieties into thedisclosed compounds (e.g., as a head-group) may further promotedisruption of the endosomal/lysosomal membrane by exploiting thefusogenicity of such amino groups. This is based not only on the pKa ofthe amino group of the composition, but also on the ability of the aminogroup to undergo a hexagonal phase transition and fuse with the vesiclemembrane. (Koltover, et al. Science (1998) 281: 78-81.) The result isbelieved to promote the disruption of the vesicle membrane and releaseof the lipid nanoparticle contents.

Similarly, in certain embodiments the incorporation of, for example, apositively charged or ionizable hydrophilic head-group in the compoundsdisclosed herein may serve to promote endosomal or lysosomal release of,for example, contents that are encapsulated in a liposomal composition(e.g., lipid nanoparticle) of the invention. Such enhanced release maybe achieved by one or both of proton-sponge mediated disruptionmechanism and/or an enhanced fusogenicity mechanism. The proton-spongemechanism is based on the ability of a compound, and in particular afunctional moiety or group of the compound, to buffer the acidificationof the endosome. This may be manipulated or otherwise controlled by thepKa of the compound or of one or more of the functional groupscomprising such compound (e.g., amino). Such endosomal disruptionproperties in turn promote osmotic swelling and the disruption of theliposomal membrane, followed by the transfection or intracellularrelease of the polynucleotide materials loaded or encapsulated thereininto the target cell.

The lipid compounds disclosed herein may generally comprise one or morecationic and/or ionizable functional head-groups, such as an aminefunctional group having one or more alkyl or aryl substituents. Incertain embodiments the lipid compounds disclosed herein may comprise acationic ionizable amino functional head-group to which is bound (e.g.,covalently bound) a hydrophobic functional groups, substituents ormoieties (e.g., an R₁ group and a R₂ group, wherein both R₁ and R₂ areindependently selected from the group consisting of hydrogen and C₁-C₁₀alkyls). In certain embodiments, such hydrophilic and hydrophobicfunctional groups are bound (e.g., covalently bound) to each other byway of an intermediary group (e.g., an alkyl or a variably unsaturatedalkenyl).

The compounds described herein (e.g., HGT5000, HGT5001 and HGT5002), arealso characterized by their reduced toxicity, in particular relative totraditional lipids and cationic lipids such as C12-200. Accordingly, oneor more of the compounds disclosed herein may be used in lieu of one ormore traditional lipids that are characterized as being toxic in theamounts necessary to deliver an effective amount of one or more agentsto target cells and tissues. For example, in some embodiments,pharmaceutical and liposomal compositions may be prepared such that theycomprise one or more of the ionizable cationic lipid compounds disclosedherein (e.g., HGT5000, HGT5001, and/or HGT5002) as a means of reducingor otherwise eliminating the toxicity associated with the liposomalcomposition. The cationic ionizable compounds or lipids (e.g., HGT5000,HGT5001 and/or HGT5002) may be used as the sole cationic lipid in one ormore of the pharmaceutical and liposomal compositions described herein(e.g., lipid nanoparticles), or alternatively may be combined withtraditional cationic lipids (e.g., LIPOFECTIN or LIPOFECTAMINE),non-cationic lipids, PEG-modified lipids and/or helper lipids. Incertain embodiments, the compounds described herein, or alternativelythe total cationic lipid component of the pharmaceutical and liposomalcompositions may comprise a molar ratio of about 1% to about 90%, about2% to about 70%, about 5% to about 50%, about 10% to about 40% of thetotal lipid present in such pharmaceutical or liposomal composition(e.g., a lipid nanoparticle), or preferably about 20% to about 70% ofthe total lipid present in such pharmaceutical or liposomal composition(e.g., a lipid nanoparticle). Additionally, combining or enrichingliposomal vehicles with the cationic ionizable lipid compounds disclosedherein allows a corresponding reduction in the concentration of theother lipid components of the liposomal vehicle, thereby providing ameans of reducing or otherwise mitigating the toxicity associated withother cationic lipids (e.g., C12-200) that may also be present in theliposomal vehicle.

In certain embodiments, at least one of the functional groups ofmoieties that comprise the compounds disclosed herein is hydrophobic innature (e.g., a hydrophobic tail-group comprising a naturally-occurringlipid such as cholesterol). As used herein, the term “hydrophobic” isused to indicate in qualitative terms that a functional group iswater-avoiding, and typically such groups are not water soluble. Forexample, in certain embodiments the hydrophobic or lipophilic tail-group(e.g., one or more of an L₁ group and an L₂ group) of the compoundsdisclosed herein may comprise one or more non-polar groups such ascholesterol or an optionally substituted, variably saturated orunsaturated alkyl or alkenyl (e.g., an optionally substitutedoctadeca-9,12-diene).

In certain embodiments, the compounds disclosed herein comprise, forexample, at least one hydrophilic head-group and at least onehydrophobic tail-group, each bound to each other by, for example anoptionally substituted, variably saturated or unsaturated alkyl oralkenyl, thereby rendering such compounds amphiphilic. As used herein todescribe a compound or composition, the term “amphiphilic” means theability to dissolve in both polar (e.g., water) and non-polar (e.g.,lipid) environments. For example, in certain embodiments, the compoundsdisclosed herein comprise at least one lipophilic tail-group (e.g.,cholesterol or a C₆-C₂₀ alkyl or alkenyl) and at least one hydrophilichead-group (e.g., an alkyl amino), each bound to an intermediary C₁-C₂₀alkyl or alkenyl group (e.g., hexane or hexene).

It should be noted that the terms “head-group” and “tail-group” as useddescribe the compounds of the present invention, and in particularfunctional groups that comprise such compounds, are used for ease ofreference to describe the orientation of one or more functional groupsrelative to other functional groups. For example, in certain embodimentsa hydrophilic head-group (e.g., amino) is bound (e.g., by one or more ofhydrogen-bonds, van der Waals' forces, ionic interactions and covalentbonds) to an alkyl or alkenyl functional group (e.g., hex-1-ene), whichin turn is bound to a hydrophobic tail-group (e.g., cholesterol or aC₆-C₂₀ variably unsaturated alkenyl).

Also disclosed herein are compounds having the structure of formula (I):

wherein R₁ and R₂ are each independently selected from the groupconsisting of hydrogen, an optionally substituted, variably saturated orunsaturated C₁-C₂₀ alkyl or alkenyl and an optionally substituted,variably saturated or unsaturated C₆-C₂₀ acyl; wherein L₁ and L₂ areeach independently selected from the group consisting of hydrogen, anoptionally substituted C₁-C₃₀ alkyl, an optionally substituted variablyunsaturated C₁-C₃₀ alkenyl, and an optionally substituted C₁-C₃₀alkynyl; wherein m and o are each independently selected from the groupconsisting of zero and any positive integer (e.g., where m is three);and wherein n is zero or any positive integer (e.g., where n is one).

In certain embodiments, the compound has the structure of formula (I),wherein R₁ and R₂ are each methyl. In such embodiment, the polarcationic head-group of the compound comprises an ionizable dimethylamino group.

In some embodiments, the compound has the structure of formula (I),wherein L₁ and L₂ are each an optionally substituted, polyunsaturatedC₆-C₂₀ alkenyl. For example, contemplated are compounds wherein L₁ andL₂ are each an optionally substituted polyunsaturated C₁₈ alkenyl. Inother embodiments, L₁ and L₂ are each an unsubstituted, polyunsaturatedC₁₈ alkenyl. In yet other embodiments, L₁ and L₂ are each an optionallysubstituted octadeca-9,12-diene (or octadec-6,9-diene). In still otherembodiments, L₁ is hydrogen and L₂ is cholesterol. In certainembodiments, each of L₁ and L₂ are (9Z,12Z)-octadeca-9,12-dien. Incertain embodiments, each of L₁ and L₂ are octadec-6,9-diene.

In certain embodiments disclosed herein, the present inventions relateto a compound having the structure of formula (I), wherein o is zero.Alternatively, in other embodiments, o is a positive integer (e.g., one,two, three, four, five, six, seven, eight, nine, ten, eleven, twelve,thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen,twenty, or more).

In certain embodiments disclosed herein, the present inventions relateto a compound having the structure of formula (I), wherein m is apositive integer (e.g., one, two, three, four, five, six, seven, eight,nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen,seventeen, eighteen, nineteen, twenty, or more). In some particularembodiments, the present inventions relate to a compound having thestructure of formula (I), wherein m is four. In some particularembodiments, the present inventions relate to a compound having thestructure of formula (I), wherein m is three.

Also disclosed herein are compounds having the structure of formula (I),wherein n is a positive integer (e.g., one, two, three, four, five, six,seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen,sixteen, seventeen, eighteen, nineteen, twenty, or more). In otherparticular embodiments, the present inventions relate to a compoundhaving the structure of formula (I), wherein n is zero.

In certain embodiments, m and o are independently selected from thegroup consisting of zero, one (such that the alkyl is ethyl), two (suchthat the alkyl is methyl), three (such that the alkyl is, for example,propyl or iso-propyl), four (such that the alkyl is, for example, butyl,iso-butyl, sec-butyl or ter-butyl), five (such that the alkyl is, forexample, pentane), six (such that the alkyl is, for example, hexane),seven (such that the alkyl is, for example, heptane), eight (such thatthe alkyl is, for example, octane), nine (n such that the alkyl is, forexample, nonane) or ten (such that the alkyl is, for example, decane).

In some particular embodiments, the present invention relates to acompound having the structure of formula (I), wherein R₁ and R₂ are eachmethyl; wherein L₁ and L₂ are each octadeca-9,12-diene (oroctadec-6,9-diene); wherein m is four; wherein n is zero; and wherein ois one. For example, in certain embodiments, the present inventionrelates to the compound(15Z,18Z)—N,N-dimethyl-6-(9Z,12Z)-octadeca-9,12-dien-1-yl)tetracosa-15,18-dien-1-amine,having the structure of formula (II), (referred to herein as “HGT5000”).

In some particular embodiments, the present invention relates to acompound having the structure of formula (I), wherein R₁ and R₂ are eachmethyl; wherein L₁ and L₂ are each octadeca-9,12-diene (oroctadec-6,9-diene); wherein m is 3; wherein n is one; and wherein o iszero. For example, in certain embodiments, the present invention relatesto the compound(15Z,18Z)—N,N-dimethyl-6-((9Z,12Z)-octadeca-9,12-dien-1-yl)tetracosa-4,15,18-trien-1-amine,having the structure of formula (III), (referred to herein as“HGT5001”).

It should be understood that in those embodiments disclosed herein wheren is one, such compounds may be a cis isomer, a trans isomer oralternatively a racemic mixture thereof. For example, in certainembodiments where n is one, n is a cis isomer, as represented by acompound having the structure of formula (IV):

Alternatively, in other embodiments where n is one, n is a trans isomer,as represented by a compound having the structure of formula (V):

Also disclosed are compounds having the structure of formula (VI):

wherein R₁ and R₂ are each independently selected from the groupconsisting of hydrogen, an optionally substituted, variably saturated orunsaturated C₁-C₂₀ alkyl or alkenyl and an optionally substituted,variably saturated or unsaturated C₆-C₂₀ acyl; wherein L₁ and L₂ areeach independently selected from the group consisting of hydrogen, anoptionally substituted C₁-C₃₀ alkyl, an optionally substituted variablyunsaturated C₁-C₃₀ alkenyl, and an optionally substituted C₁-C₃₀alkynyl; and wherein m, n and o are each independently selected from thegroup consisting of zero and any positive integer.

In some particular embodiments, the present inventions are directed to acompound having the structure of formula (VI), wherein R₁ and R₂ areeach methyl. In other embodiments, the present inventions are directedto a compound having the structure of formula (VI), wherein R₁ and R₂are each independently selected from the group consisting of hydrogenand methyl.

Also contemplated are compounds having the structure of formula (VI),wherein L₁ and L₂ are each an optionally substituted, polyunsaturatedC₆-C₂₀ alkenyl (e.g., where L₁ and L₂ are each an optionally substitutedpolyunsaturated C₁₈ alkenyl or where L₁ and L₂ are each anunsubstituted, polyunsaturated C₁₈ alkenyl). In certain embodiments,disclosed herein, L₁ and L₂ are each an optionally substitutedoctadeca-9,12-diene (or octadec-6,9-diene). In other embodiments L₁ ishydrogen and L₂ is cholesterol.

In certain embodiments disclosed herein, the present inventions relateto a compound having the structure of formula (VI), wherein m is apositive integer (e.g., one, two, three, four, five, six, seven, eight,nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen,seventeen, eighteen, nineteen, twenty, or more). In some particularembodiments, the present inventions relate to a compound having thestructure of formula (VI), wherein m is four. In certain embodiments,the present inventions relate to a compound having the structure offormula (VI), wherein m is at least five (e.g., where m is five, six,seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen,sixteen, seventeen, eighteen, nineteen, twenty or more).

Also disclosed herein are compounds having the structure of formula(VI), wherein n is a positive integer (e.g., one, two, three, four,five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen,fifteen, sixteen, seventeen, eighteen, nineteen, twenty, or more). Inother particular embodiments, the present inventions relate to acompound having the structure of formula (VI), wherein n is zero.

In certain embodiments disclosed herein, the present inventions aredirected to compounds having the structure of formula (VI), wherein o isa positive integer (e.g., one, two, three, four, five, six, seven,eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen,seventeen, eighteen, nineteen, twenty, or more). In certain embodiments,the present inventions relate to a compound having the structure offormula (VI), wherein o is at least five (e.g., where o is five, six,seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen,sixteen, seventeen, eighteen, nineteen, twenty or more). Alternatively,in other particular embodiments, the present inventions relate tocompounds having the structure of formula (VI), wherein o is zero.

Also contemplated are compounds having the structure of formula (VI),wherein R₁ and R₂ are each methyl; wherein L₁ and L₂ are eachoctadeca-9,12-diene (or octadec-6,9-diene); wherein m is 4; and whereinboth n and o are zero. For example, in certain embodiments, the presentinvention relates to the compound(15Z,18Z)—N,N-dimethyl-6-((9Z,12Z)-octadeca-9,12-dien-1-yl)tetracosa-5,15,18-trien-1-aminehaving the structure of formula (VII), (referred to herein as“HGT5002”):

Also disclosed herein are compounds having the structure of formula(VIII):

wherein R₁ and R₂ are each independently selected from the groupconsisting of an optionally substituted, variably saturated orunsaturated C₁-C₂₀ alkyl or alkenyl and an optionally substituted,variably saturated or unsaturated C₆-C₂₀ acyl; wherein L₁ and L₂ areeach independently selected from the group consisting of an optionallysubstituted C₁-C₃₀ alkyl, an optionally substituted variably unsaturatedC₁-C₃₀ alkenyl, and an optionally substituted C₁-C₃₀ alkynyl; andwherein x is selected from the group consisting of a C₁-C₂₀ alkyl and avariably unsaturated C₁-C₂₀ alkenyl.

In certain embodiments, the disclosed compounds have the structure offormula (VIII), wherein R₁ and R₂ are each methyl. In other embodiments,the disclosed compounds have the structure of formula (VIII), wherein R₁and R₂ are independently selected from the group consisting of hydrogenand a C₁-C₆ alkyl.

In other embodiments, the present invention relates to compounds havingthe structure of formula (VIII), wherein L₁ and L₂ are each anunsubstituted, polyunsaturated C₁₈ alkenyl. For example, in certainembodiments, L₁ and L₂ are each an optionally substitutedoctadeca-9,12-diene (e.g., L₁ and L₂ are each an unsubstitutedoctadeca-9,12-diene or octadec-6,9-diene). In certain other embodiments,L₁ is hydrogen and L₂ is cholesterol.

In certain embodiments, the disclosed compounds have the structure offormula (VIII), wherein x is a C₆ alkenyl. In other embodiments, thedisclosed compounds have the structure of formula (VIII), wherein x ishexane. In yet other embodiments, the disclosed compounds have thestructure of formula (VIII), wherein x is hex-1-ene. In still otherembodiments, the disclosed compounds have the structure of formula(VIII), wherein x is hex-2-ene. In certain embodiments, x is not hexane.In other embodiments, the disclosed compounds have the structure offormula (VIII), wherein x is a C₆-C₁₀ alkenyl or a C₆-C₁₀ alkyl.

In one particular embodiment, the present invention relates to acompound having the structure of formula (VIII), wherein R₁ and R₂ areeach methyl; wherein L₁ and L₂ are each octadeca-9,12-diene; and whereinx is hexane. In another particular embodiment, the present inventionrelates to a compound having the structure of formula (VIII), wherein R₁and R₂ are each methyl; wherein L₁ and L₂ are each octadeca-9,12-diene(or octadec-6,9-diene); and wherein x is hex-1-ene. In still anotherparticular embodiment, the present invention relates to a compoundhaving the structure of formula (VIII), wherein R₁ and R₂ are eachmethyl; wherein L₁ and L₂ are each octadeca-9,12-diene (oroctadec-6,9-diene); and wherein x is hex-2-ene.

As used herein, the term “alkyl” refers to both straight and branchedchain C₁-C₄₀ hydrocarbons (e.g., C₆-C₂₀ hydrocarbons), and include bothsaturated and unsaturated hydrocarbons. In certain embodiments, thealkyl may comprise one or more cyclic alkyls and/or one or moreheteroatoms such as oxygen, nitrogen, or sulfur and may optionally besubstituted with substituents (e.g., one or more of alkyl, halo,alkoxyl, hydroxy, amino, aryl, ether, ester or amide). In certainembodiments, a contemplated alkyl hydrophobic tail-group comprises(9Z,12Z)-octadeca-9,12-dien. In certain embodiments, a contemplatedalkyl hydrophobic tail-group comprises (or octadec-6,9-diene. The use ofdesignations such as, for example, “C₆-C₂₀” is intended to refer to analkyl (e.g., straight or branched chain and inclusive of alkenes andalkyls) having the recited range carbon atoms.

As used herein, the term “aryl” refers to aromatic groups (e.g.,monocyclic, bicyclic and tricyclic structures) containing six to tencarbons in the ring portion. The aryl groups may be optionallysubstituted through available carbon atoms and in certain embodimentsmay include one or more heteroatoms such as oxygen, nitrogen or sulfur.

It should be understood that in those embodiments described herein wherethe compounds have one or more asymmetric or chiral molecules (e.g., oneor more unsaturated carbon-carbon double bonds), both the cis (Z) andtrans (E) isomers are within the scope of this invention.

The compounds described herein may be used to construct liposomalcompositions that facilitate or enhance the delivery and release ofencapsulated materials (e.g., one or more therapeutic polynucleotides)to one or more target cells (e.g., by permeating or fusing with thelipid membranes of such target cells). For example, when a liposomalcomposition (e.g., a lipid nanoparticle) comprises or is otherwiseenriched with one or more of the compounds disclosed herein, the phasetransition in the lipid bilayer of the one or more target cells mayfacilitate the delivery of the encapsulated materials (e.g., one or moretherapeutic polynucleotides encapsulated in a lipid nanoparticle) intothe one or more target cells. Similarly, in certain embodiments thecompounds disclosed herein may be used to prepare liposomal vehiclesthat are characterized by their reduced toxicity in vivo. In certainembodiments, the reduced toxicity is a function of the high transfectionefficiencies associated with the compositions disclosed herein, suchthat a reduced quantity of such composition may administered to thesubject to achieve a desired therapeutic response or outcome.

In certain embodiments the compounds described herein are characterizedas having one or more properties that afford such compounds advantagesrelative to other similarly classified lipids. For example, in certainembodiments, the compounds disclosed herein allow for the control andtailoring of the properties of liposomal compositions (e.g., lipidnanoparticles) of which they are a component. In particular, thecompounds disclosed herein may be characterized by enhanced transfectionefficiencies and their ability to provoke specific biological outcomes.Such outcomes may include, for example enhanced cellular uptake,endosomal/lysosomal disruption capabilities and/or promoting the releaseof encapsulated materials (e.g., polynucleotides) intracellularly.

In certain embodiments the compounds described herein (and thepharmaceutical and liposomal compositions comprising such compounds)employ a multifunctional strategy to facilitate the delivery ofencapsulated materials (e.g., one or more polynucleotides) to, andsubsequent transfection of one or more target cells. For example, incertain embodiments the compounds described herein (and thepharmaceutical and liposomal compositions comprising such compounds) arecharacterized as having one or more of receptor-mediated endocytosis,clathrin-mediated and caveolae-mediated endocytosis, phagocytosis andmacropinocytosis, fusogenicity, endosomal or lysosomal disruption and/orreleasable properties that afford such compounds advantages relativeother similarly classified lipids.

In certain embodiments the compounds and the pharmaceutical andliposomal compositions of which such compounds are a component (e.g.,lipid nanoparticles) exhibit an enhanced (e.g., increased) ability totransfect one or more target cells. Accordingly, also provided hereinare methods of transfecting one or more target cells. Such methodsgenerally comprise the step of contacting the one or more target cellswith the compounds and/or pharmaceutical compositions disclosed herein(e.g., an HGT5000-, HGT5001- and/or HGT5002-based lipid nanoparticleencapsulating one or more polynucleotides) such that the one or moretarget cells are transfected with the materials encapsulated therein(e.g., one or more polynucleotides). As used herein, the terms“transfect” or “transfection” refer to the intracellular introduction ofone or more encapsulated materials (e.g., nucleic acids and/orpolynucleotides) into a cell, or preferably into a target cell. Theintroduced polynucleotide may be stably or transiently maintained in thetarget cell. The term “transfection efficiency” refers to the relativeamount of such encapsulated material (e.g., polynucleotides) up-takenby, introduced into and/or expressed by the target cell which is subjectto transfection. In practice, transfection efficiency may be estimatedby the amount of a reporter polynucleotide product produced by thetarget cells following transfection. In certain embodiments, thecompounds and pharmaceutical compositions described herein demonstratehigh transfection efficiencies thereby improving the likelihood thatappropriate dosages of the encapsulated materials (e.g., one or morepolynucleotides) will be delivered to the site of pathology andsubsequently expressed, while at the same time minimizing potentialsystemic adverse effects or toxicity associated with the compound ortheir encapsulated contents.

A wide range of materials that can exert pharmaceutical or therapeuticeffects can be delivered to target cells using the compounds,compositions and methods of the present invention. Accordingly, thecompounds and pharmaceutical and liposomal compositions described hereinmay be used to encapsulate any materials suitable for intracellulardelivery. In certain embodiments, such encapsulated materials arecapable of conferring a therapeutic or diagnostic benefit upon the cellsinto which such materials are delivered, and may include any drugs,biologics and/or diagnostics. The materials can be organic or inorganic.Organic molecules can be peptides, proteins, carbohydrates, lipids,sterols, nucleic acids (including peptide nucleic acids), or anycombination thereof. In certain embodiments, the pharmaceutical andliposomal compositions described herein can comprise or otherwiseencapsulate more than one type of material, for example, two or moredifferent polynucleotide sequences encoding a protein, an enzyme and/ora steroid. In certain embodiments, the encapsulated materials are one ormore polynucleotides and nucleic acids.

As used herein, the terms “polynucleotide” and “nucleic acid” are usedinterchangeably to refer to genetic material (e.g., DNA or RNA), andwhen such terms are used with respect to the compounds and compositionsdescribed herein (e.g., lipid nanoparticles) generally refer to thegenetic material encapsulated by such compounds and compositions (e.g.,lipid nanoparticles). In some embodiments, the polynucleotide is RNA.Suitable RNA includes mRNA, siRNA, miRNA, snRNA and snoRNA. Contemplatedpolynucleotides also include large intergenic non-coding RNA (lincRNA),which generally does not encode proteins, but rather function, forexample, in immune signaling, stem cell biology and the development ofdisease. (See, e.g., Guttman, et al., 458: 223-227 (2009); and Ng, etal., Nature Genetics 42: 1035-1036 (2010), the contents of which areincorporated herein by reference). In certain embodiments, thepolynucleotides encapsulated by the compounds or pharmaceutical andliposomal compositions of the invention include RNA or stabilized RNAencoding a protein or enzyme (e.g., mRNA encoding α-galactosidase A orarylsulfatase A). The present invention contemplates the use of suchpolynucleotides (and in particular RNA or stabilized RNA) as atherapeutic that is capable of being expressed by target cells tothereby facilitate the production (and in certain instances theexcretion) of a functional enzyme or protein by such target cells asdisclosed for example, in International Application No.PCT/US2010/058457 and in U.S. Provisional Application No. 61/494,881,filed Jun. 8, 2011, the teachings of which are both incorporated hereinby reference in their entirety. For example, in certain embodiments,upon the expression of one or more polynucleotides by target cells theproduction of a functional enzyme or protein in which a subject isdeficient (e.g., a urea cycle enzyme or an enzyme associated with alysosomal storage disorder) may be observed. The term “functional”, asused herein to qualify a protein or enzyme, means that the protein orenzyme has biological activity, or alternatively is able to perform thesame, or a similar function as the native or normally-functioningprotein or enzyme.

In certain embodiments, the compounds and the pharmaceutical andliposomal compositions described herein are formulated as a blendedformulation or composition. For example, in one embodiment, apharmaceutical composition comprises a blended formulation comprising a3:1 ratio of a first lipid nanoparticle comprising HGT5000 and a secondlipid nanoparticle comprising HGT5001. Accordingly, also provided hereinare blended pharmaceutical compositions and related methods formodulating the expression of a polynucleotide in one or more targetcells and tissues, as disclosed for example, in U.S. ProvisionalApplication No. 61/494,714, filed Jun. 8, 2011, the teachings of whichare incorporated herein by reference in their entirety. Alsocontemplated are methods for modulating (e.g., increasing orsynergistically increasing) the production and/or secretion of, forexample, one or more functional polypeptides, proteins or enzymes thatare encoded by one or more polynucleotides (e.g., mRNA) encapsulated insuch blended pharmaceutical compositions by one or more target cells.

In the context of the present invention the term “expression” is used inits broadest sense to refer to either the transcription of a specificgene or polynucleotide into at least one mRNA transcript, or thetranslation of at least one mRNA or polynucleotide into a protein orenzyme. For example, in certain embodiments the compounds and thepharmaceutical or liposomal compositions described herein comprise apolynucleotide (e.g., mRNA) which encodes a functional protein orenzyme. In the context of such mRNA polynucleotides, the term expressionrefers to the translation of such mRNA (e.g., by the target cells) toproduce the polypeptide or protein encoded thereby.

In certain embodiments, the compounds and pharmaceutical compositionsprovided herein are capable of modulating the expression of aberrantlyexpressed nucleic acids and polynucleotides in one or more target cellsand tissues. Accordingly, also provided herein are methods of treatingdisease in a subject by administering an effective amount of thecompounds and/or the pharmaceutical or liposomal compositions describedherein to the subject. In certain embodiments, such methods may enhance(e.g., increase) the expression of a polynucleotide and/or increase theproduction and secretion of a functional polypeptide product in one ormore target cells and tissues (e.g., hepatocytes). In some embodiments,the targeted cells or tissues aberrantly express the polynucleotideencapsulated by one or more of the compounds or pharmaceutical andliposomal compositions (e.g., lipid nanoparticles) described herein.Also provided herein are methods of increasing the expression of one ormore polynucleotides (e.g., mRNA) in one or more target cells, tissuesand organs (e.g., the lungs, heart, spleen, liver and/or kidneys).Generally, such methods comprise contacting the target cells with one ormore compounds and/or pharmaceutical or liposomal compositions thatcomprise or otherwise encapsulate one or more polynucleotides.

In certain embodiments, the compounds disclosed herein may be used as aliposome or as a component of a liposome. Specifically, in certainembodiments the compounds disclosed herein may be used as a lipid (e.g.,cationic lipid) component of a liposomal composition (e.g., a lipidnanoparticle). Such liposomes may be used to encapsulate materials andfacilitate the delivery of such materials to one or more target cells,tissues and organs. As used herein, the term “liposome” generally refersto a vesicle composed of lipids (e.g., amphiphilic lipids) arranged inone or more spherical bilayer or bilayers. In certain embodiments, theliposome is a lipid nanoparticle (e.g., a lipid nanoparticle comprisingone or more of the cationic lipid compounds disclosed herein). Suchliposomes may be unilamellar or multilamellar vesicles which have amembrane formed from a lipophilic material and an aqueous interior thatcontains the encapsulated materials (e.g., polynucleotides) to bedelivered to one or more target cells, tissues and organs. In certainembodiments, the pharmaceutical and liposomal compositions describedherein comprise one or more lipid nanoparticles. Contemplated liposomesinclude lipid nanoparticles. Examples of suitable lipids (e.g., cationiclipids) that may be used to form the liposomes and lipid nanoparticlescontemplated hereby include one or more of the compounds disclosedherein (e.g., HGT5000, HGT5001, and/or HGT5002). Such liposomes andlipid nanoparticles may also comprise additional cationic lipids such asC12-200, DLin-KC2-DMA, DOPE, DMG-PEG-2000, non-cationic lipids,cholesterol-based lipids, helper lipids, PEG-modified lipids, as well asthe phosphatidyl compounds (e.g., phosphatidylglycerol,phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine,sphingolipids, cerebrosides, and gangliosides) and combinations ormixtures of the forgoing.

Several cationic lipids have been described in the literature, many ofwhich are commercially available. In certain embodiments, such cationiclipids are included in the pharmaceutical or liposomal compositionsdescribed herein in addition to one or more of the compounds or lipidsdisclosed herein (e.g., HGT5000). In some embodiments, the cationiclipid N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride or“DOTMA” is used. (Felgner et al. (Proc. Nat'l Acad. Sci. 84, 7413(1987); U.S. Pat. No. 4,897,355). DOTMA can be formulated alone or canbe combined with dioleoylphosphatidylethanolamine or “DOPE” or othercationic or non-cationic lipids into a lipid nanoparticle. Othersuitable cationic lipids include, for example C12-200,5-carboxyspermylglycinedioctadecylamide or “DOGS,”2,3-dioleyloxy-N-[2(spermine-carboxamido)ethyl]-N,N-dimethyl-1-propanaminiumor “DOSPA” (Behr et al. Proc. Nat.'l Acad. Sci. 86, 6982 (1989); U.S.Pat. Nos. 5,171,678; 5,334,761), 1,2-Dioleoyl-3-Dimethylammonium-Propaneor “DODAP”, 1,2-Dioleoyl-3-Trimethylammonium-Propane or “DOTAP”.Contemplated cationic lipids also include1,2-distearyloxy-N,N-dimethyl-3-aminopropane or “DSDMA”,1,2-dioleyloxy-N,N-dimethyl-3-aminopropane or “DODMA”,1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane or “DLinDMA”,1,2-dilinolenyloxy-N,N-dimethyl-3-aminopropane or “DLenDMA”,N-dioleyl-N,N-dimethylammonium chloride or “DODAC”,N,N-distearyl-N,N-dimethylammonium bromide or “DDAB”,N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammoniumbromide or “DMRIE”,3-dimethylamino-2-(cholest-5-en-3-beta-oxybutan-4-oxy)-1-(cis,cis-9,12-octadecadienoxy)propaneor “CLinDMA”,2-[5′-(cholest-5-en-3-beta-oxy)-3′-oxapentoxy)-3-dimethyl-1-(cis,cis-9′,1-2′-octadecadienoxy)propaneor “CpLinDMA”, N,N-dimethyl-3,4-dioleyloxybenzylamine or “DMOBA”,1,2-N,N′-dioleylcarbamyl-3-dimethylaminopropane or “DOcarbDAP”,2,3-Dilinoleoyloxy-N,N-dimethylpropylamine or “DLinDAP”,1,2-N,N′-Dilinoleylcarbamyl-3-dimethylaminopropane or “DLincarbDAP”,1,2-Dilinoleoylcarbamyl-3-dimethylaminopropane or “DLinCDAP”,2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane or “DLin-K-DMA”,2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane or“DLin-K-XTC2-DMA”, or mixtures thereof (Heyes, J., et al., J ControlledRelease 107: 276-287 (2005); Morrissey, D V., et al., Nat. Biotechnol.23(8): 1003-1007 (2005); PCT Publication WO2005/121348A1). The use ofcholesterol-based cationic lipids to formulate the compositions (e.g.,lipid nanoparticles) is also contemplated by the present invention. Suchcholesterol-based cationic lipids can be used, either alone or incombination with other cationic or non-cationic lipids. Suitablecholesterol-based cationic lipids include, for example, DC-Chol(N,N-dimethyl-N-ethylcarboxamidocholesterol),1,4-bis(3-N-oleylamino-propyl)piperazine (Gao, et al. Biochem. Biophys.Res. Comm. 179, 280 (1991); Wolf et al. BioTechniques 23, 139 (1997);U.S. Pat. No. 5,744,335).

In addition, several reagents are commercially available to enhancetransfection efficacy. Suitable examples include LIPOFECTIN (DOTMA:DOPE)(Invitrogen, Carlsbad, Calif.), LIPOFECTAMINE (DOSPA:DOPE) (Invitrogen),LIPOFECTAMINE2000. (Invitrogen), FUGENE, TRANSFECTAM (DOGS), andEFFECTENE. Also contemplated are cationic lipids such as thedialkylamino-based, imidazole-based, and guanidinium-based lipids. Forexample, also contemplated is the use of the cationic lipid(3S,10R,13R,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl3-(1H-imidazol-4-yl)propanoate or “ICE”, as disclosed in InternationalApplication No. PCT/US2010/058457, the teachings of which areincorporated herein by reference in their entirety.

The use and inclusion of polyethylene glycol (PEG)-modifiedphospholipids and derivatized lipids such as derivatized cerarmides(PEG-CER), including N-Octanoyl-Sphingosine-1-[Succinyl(MethoxyPolyethylene Glycol)-2000] (C8 PEG-2000 ceramide) in the liposomal andpharmaceutical compositions described herein is also contemplated,preferably in combination with one or more of the compounds and lipidsdisclosed herein. Contemplated PEG-modified lipids include, but are notlimited to, a polyethylene glycol chain of up to 5 kDa in lengthcovalently attached to a lipid with alkyl chain(s) of C₆-C₂₀ length. Theaddition of such components may prevent complex aggregation and may alsoprovide a means for increasing circulation lifetime and increasing thedelivery of the lipid-polynucleotide composition to the target tissues,(Klibanov et al. (1990) FEBS Letters, 268 (1): 235-237), or they may beselected to rapidly exchange out of the formulation in vivo (see U.S.Pat. No. 5,885,613). Particularly useful exchangeable lipids arePEG-ceramides having shorter acyl chains (e.g., C14 or C18). ThePEG-modified phospholipid and derivatized lipids of the presentinvention may comprise a molar ratio from about 0% to about 20%, about0.5% to about 20%, about 1% to about 15%, about 4% to about 10%, orabout 2% of the total lipid present in a liposomal lipid nanoparticle.

The present invention also contemplates the use of non-cationic lipidsin one or more of the pharmaceutical or liposomal compositions (e.g.,lipid nanoparticles). Such non-cationic lipids are preferably used incombination with one or more of the compounds and lipids disclosedherein. As used herein, the phrase “non-cationic lipid” refers to anyneutral, zwitterionic or anionic lipid. As used herein, the phrase“anionic lipid” refers to any of a number of lipid species that carry anet negative charge at a selected pH, such as physiological pH.Non-cationic lipids include, but are not limited to,distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine(DOPC), dipalmitoylphosphatidylcholine (DPPC),dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol(DPPG), dioleoylphosphatidylethanolamine (DOPE),palmitoyloleoylphosphatidylcholine (POPC),palmitoyloleoyl-phosphatidylethanolamine (POPE),dioleoyl-phosphatidylethanolamine4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE),distearoyl-phosphatidyl-ethanolamine (DSPE), DLPE(1,2-dilauroyl-sn-glycero-3-phosphoethanolamine), DPPS(1,2-dipalmitoyl-sn-glycero-3-phospho-L-serine), 16-O-monomethyl PE,16-O-dimethyl PE, 18-1-trans PE,1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), ceramides,sphingomyelins, cholesterol, or a mixture thereof. Such non-cationiclipids may be used alone, but are preferably used in combination withother excipients, for example, one or more of the cationic lipidcompounds disclosed herein (e.g., HGT5000, HGT5001, and/or HGT5002).When used in combination with a cationic lipid, the non-cationic lipidmay comprise a molar ratio of 5% to about 90%, or preferably about 10%to about 70% of the total lipid present in the lipid nanoparticle.

Also contemplated is inclusion of polymers in the lipid nanoparticlesthat comprise the pharmaceutical or liposomal compositions describedherein. Suitable polymers may include, for example, polyacrylates,polyalkycyanoacrylates, polylactide, polylactide-polyglycolidecopolymers, polycaprolactones, dextran, albumin, gelatin, alginate,collagen, chitosan, cyclodextrins and polyethylenimine. Such polymersmay be used alone, but are preferably used in combination with otherexcipients, for example, one or more of the cationic lipid compoundsdisclosed herein (e.g., HGT5000, HGT5001, and/or HGT5002).

In certain embodiments, the pharmaceutical and liposomal compositions(e.g., lipid nanoparticles) are formulated based in part upon theirability to facilitate the transfection (e.g., of a polynucleotide) of atarget cell. In another embodiment, the pharmaceutical and liposomalcompositions (e.g., lipid nanoparticles) may be selected and/or preparedto optimize delivery of polynucleotides to a target cell, tissue ororgan. For example, if the target cell is a hepatocyte the properties ofthe pharmaceutical and/or liposomal compositions (e.g., size, chargeand/or pH) may be optimized to effectively deliver such composition(e.g., lipid nanoparticles) to the target cell or organ, reduce immuneclearance and/or promote retention in that target organ. Alternatively,if the target tissue is the central nervous system the selection andpreparation of the pharmaceutical and liposomal compositions mustconsider penetration of, and retention within the blood brain barrierand/or the use of alternate means of directly delivering suchcompositions (e.g., lipid nanoparticles) to such target tissue (e.g.,via intracerebrovascular administration). In certain embodiments, thepharmaceutical or liposomal compositions or their constituent lipidnanoparticles may be combined with agents that facilitate the transferof encapsulated materials (e.g., agents which disrupt or improve thepermeability of the blood brain barrier and thereby enhance the transferof such encapsulated polynucleotides to the target cells). While thepharmaceutical and liposomal compositions described herein (e.g., lipidnanoparticles) can facilitate introduction of encapsulated materialssuch as one or more polynucleotides into target cells, the addition ofpolycations (e.g., poly L-lysine and protamine) to, for example one ormore of the lipid nanoparticles that comprise the pharmaceuticalcompositions as a copolymer can also facilitate, and in some instancesmarkedly enhance the transfection efficiency of several types ofcationic liposomes by 2-28 fold in a number of cell lines both in vitroand in vivo. (See, N. J. Caplen, et al., Gene Ther. 1995; 2: 603; S. Li,et al., Gene Ther. 1997; 4, 891.)

In certain embodiments of the present invention, the pharmaceutical andliposomal compositions (e.g., lipid nanoparticles) are prepared toencapsulate one or more materials or therapeutic agents (e.g.,polynucleotides). The process of incorporating a desired therapeuticagent (e.g., mRNA) into a liposome or a lipid nanoparticle is referredto herein as or “loading” or “encapsulating” (Lasic, et al., FEBS Lett.,312: 255-258, 1992). The lipid nanoparticle-loaded or -encapsulatedmaterials (e.g., polynucleotides) may be completely or partially locatedin the interior space of the lipid nanoparticle, within the bilayermembrane of the lipid nanoparticle, or associated with the exteriorsurface of the lipid nanoparticle.

Loading or encapsulating, for example, a polynucleotide into a lipidnanoparticle may serve to protect the polynucleotide from an environmentwhich may contain enzymes or chemicals (e.g., serum) that degrade suchpolynucleotides and/or systems or receptors that cause the rapidexcretion of such polynucleotides. Accordingly, in some embodiments, thecompositions described herein are capable of enhancing the stability ofthe polynucleotide(s) encapsulated thereby, particularly with respect tothe environments into which such polynucleotides will be exposed.Encapsulating materials, such as for example polynucleotides into one ormore of the pharmaceutical and liposomal compositions described herein(e.g., lipid nanoparticles) also facilitates the delivery of suchpolynucleotides into the target cells and tissues. For example, lipidnanoparticles comprising one or more of the lipid compounds describedherein can allow the encapsulated polynucleotide to reach the targetcell or may preferentially allow the encapsulated polynucleotide toreach the target cells or organs on a discriminatory basis (e.g., thelipid nanoparticles may concentrate in the liver or spleens of a subjectto which such lipid nanoparticles are administered). Alternatively, thelipid nanoparticles may limit the delivery of encapsulatedpolynucleotides to other non-targeted cells or organs where the presenceof the encapsulated polynucleotides may be undesirable or of limitedutility.

In certain embodiments, the pharmaceutical and liposomal compositionsdescribed herein (e.g., lipid nanoparticles) are prepared by combiningmultiple lipid components (e.g., one or more of the compounds disclosedherein) with one or more polymer components. For example, a lipidnanoparticle may be prepared using HGT5000, DOPE, CHOL and DMG-PEG2000.A lipid nanoparticle may be comprised of additional lipid combinationsin various ratios, including for example, HGT5001, DOPE and DMG-PEG2000.The selection of cationic lipids, non-cationic lipids and/orPEG-modified lipids which comprise the lipid nanoparticles, as well asthe relative molar ratio of such lipids to each other, is based upon thecharacteristics of the selected lipid(s), the nature of the intendedtarget cells or tissues and the characteristics of the materials orpolynucleotides to be delivered by the lipid nanoparticle. Additionalconsiderations include, for example, the saturation of the alkyl chain,as well as the size, charge, pH, pKa, fusogenicity and toxicity of theselected lipid(s).

The pharmaceutical and liposomal composition (e.g., lipid nanoparticles)for use in the present invention can be prepared by various techniqueswhich are presently known in the art. Multi-lamellar vesicles (MLV) maybe prepared conventional techniques, for example, by depositing aselected lipid on the inside wall of a suitable container or vessel bydissolving the lipid in an appropriate solvent, and then evaporating thesolvent to leave a thin film on the inside of the vessel or by spraydrying. An aqueous phase may then added to the vessel with a vortexingmotion which results in the formation of MLVs. Unilamellar vesicles(ULV) can then be formed by homogenization, sonication or extrusion ofthe multi-lamellar vesicles. In addition, unilamellar vesicles can beformed by detergent removal techniques.

In certain embodiments, the pharmaceutical and liposomal compositions ofthe present invention comprise a lipid nanoparticle wherein theencapsulated polynucleotide (e.g., mRNA) is associated on both thesurface of the lipid nanoparticle and encapsulated within the same lipidnanoparticle. For example, during preparation of the compositions of thepresent invention, one or more of the cationic lipid compounds describedherein and which comprise the lipid nanoparticles may associate with thepolynucleotides (e.g., mRNA) through electrostatic interactions withsuch polynucleotides.

In certain embodiments, the pharmaceutical and liposomal compositions ofthe present invention may be loaded with diagnostic radionuclide,fluorescent materials or other materials that are detectable in both invitro and in vivo applications. For example, suitable diagnosticmaterials for use in the present invention may includeRhodamine-dioleoylphosphatidylethanolamine (Rh-PE), Green FluorescentProtein mRNA, Renilla Luciferase mRNA and Firefly Luciferase mRNA.

During the preparation of liposomal compositions described herein, watersoluble carrier agents may be also encapsulated in the aqueous interiorby including them in the hydrating solution, and lipophilic moleculesmay be incorporated into the lipid bilayer by inclusion in the lipidformulation. In the case of certain molecules (e.g., cationic or anioniclipophilic polynucleotides), loading of the polynucleotide intopreformed lipid nanoparticles or liposomes may be accomplished, forexample, by the methods described in U.S. Pat. No. 4,946,683, thedisclosure of which is incorporated herein by reference. Followingencapsulation of the polynucleotide, the lipid nanoparticles may beprocessed to remove un-encapsulated mRNA through processes such as gelchromatography, diafiltration or ultrafiltration. For example, if it isdesirous to remove externally bound polynucleotide from the surface ofthe liposomal compositions (e.g., lipid nanoparticles) described herein,such lipid nanoparticles may be subject to a Diethylaminoethyl SEPHACELcolumn.

In addition to the encapsulated materials (e.g., polynucleotides or oneor more therapeutic or diagnostic agents) may be included orencapsulated in the lipid nanoparticle. For example, such additionaltherapeutic agents may be associated with the surface of the lipidnanoparticle, can be incorporated into the lipid bilayer of the lipidnanoparticle by inclusion in the lipid formulation or loading intopreformed lipid nanoparticles (See, U.S. Pat. Nos. 5,194,654 and5,223,263, which are incorporated by reference herein).

There are several methods for reducing the size, or “sizing”, of theliposomal compositions (e.g., lipid nanoparticles) disclosed herein, andany of these methods may generally be employed when sizing is used aspart of the invention. The extrusion method is a one method of liposomesizing. (Hope, M J et al. Reduction of Liposome Size and Preparation ofUnilamellar Vesicles by Extrusion Techniques. In: Liposome Technology(G. Gregoriadis, Ed.) Vol. 1. p 123 (1993)). The method consists ofextruding liposomes through a small-pore polycarbonate membrane or anasymmetric ceramic membrane to reduce liposome sizes to a relativelywell-defined size distribution. Typically, the suspension is cycledthrough the membrane one or more times until the desired liposome sizedistribution is achieved. The liposomes may be extruded throughsuccessively smaller pore membranes to achieve gradual reduction inliposome size.

A variety of alternative methods known in the art are available forsizing of a population of lipid nanoparticles. One such sizing method isdescribed in U.S. Pat. No. 4,737,323, incorporated herein by reference.Sonicating a liposome or lipid nanoparticle suspension either by bath orprobe sonication produces a progressive size reduction down to small ULVless than about 0.05 microns in diameter. Homogenization is anothermethod that relies on shearing energy to fragment large liposomes intosmaller ones. In a typical homogenization procedure, MLV arerecirculated through a standard emulsion homogenizer until selectedliposome sizes, typically between about 0.1 and 0.5 microns, areobserved. The size of the lipid nanoparticles may be determined byquasi-electric light scattering (QELS) as described in Bloomfield, Ann.Rev. Biophys. Bioeng., 10:421-450 (1981), incorporated herein byreference. Average lipid nanoparticle diameter may be reduced bysonication of formed lipid nanoparticles. Intermittent sonication cyclesmay be alternated with QELS assessment to guide efficient liposomesynthesis.

Selection of the appropriate size of the liposomal compositionsdescribed herein (e.g., lipid nanoparticles) must take intoconsideration the site of the target cell or tissue and to some extentthe application for which the lipid nanoparticle is being made. As usedherein, the phrase “target cell” refers to cells to which one or more ofthe pharmaceutical and liposomal compositions described herein are to bedirected or targeted. In some embodiments, the target cells comprise aparticular tissue or organ. In some embodiments, the target cells aredeficient in a protein or enzyme of interest. For example, where it isdesired to deliver a polynucleotide to a hepatocyte, the hepatocyterepresents the target cell. In some embodiments, the pharmaceutical orliposomal compositions (and for example the polynucleotide materialsencapsulated therein) of the present invention transfect the targetcells on a discriminatory basis (i.e., do not transfect non-targetcells). The compositions and methods of the present invention may beprepared to preferentially target a variety of target cells, whichinclude, but are not limited to, hepatocytes, hematopoietic cells,epithelial cells, endothelial cells, lung cells, alveolar cells, bonecells, stem cells, mesenchymal cells, neural cells (e.g., meninges,astrocytes, motor neurons, cells of the dorsal root ganglia and anteriorhorn motor neurons), photoreceptor cells (e.g., rods and cones), retinalpigmented epithelial cells, secretory cells, cardiac cells, adipocytes,vascular smooth muscle cells, cardiomyocytes, skeletal muscle cells,beta cells, pituitary cells, synovial lining cells, ovarian cells,testicular cells, fibroblasts, B cells, T cells, reticulocytes,leukocytes, granulocytes and tumor cells.

Following transfection of one or more target cells by, for example, thepolynucleotides encapsulated in the one or more lipid nanoparticlescomprising the pharmaceutical or liposomal compositions disclosedherein, the production of the product (e.g., a polypeptide or protein)encoded by such polynucleotide may be preferably stimulated and thecapability of such target cells to express the polynucleotide andproduce, for example, a polypeptide or protein of interest is enhanced.For example, transfection of a target cell by one or more compounds orpharmaceutical compositions encapsulating mRNA will enhance (i.e.,increase) the production of the protein or enzyme encoded by such mRNA.

In some embodiments, it may be desirable to limit transfection of thepolynucleotides to certain cells or tissues. For example, the liverrepresents an important target organ for the compositions of the presentinvention in part due to its central role in metabolism and productionof proteins and accordingly diseases which are caused by defects inliver-specific gene products (e.g., the urea cycle disorders) maybenefit from specific targeting of cells (e.g., hepatocytes).Accordingly, in certain embodiments of the present invention, thestructural characteristics of the target tissue may be exploited todirect the distribution of the pharmaceutical and liposomal compositionsof the present invention (e.g., an HGT5001-based lipid nanoparticle) tosuch target tissues. For example, to target hepatocytes one or more ofthe lipid nanoparticles that comprise the pharmaceutical or liposomalcompositions described herein may be sized such that their dimensionsare smaller than the fenestrations of the endothelial layer lininghepatic sinusoids in the liver; accordingly the one or more of suchlipid nanoparticles can readily penetrate such endothelial fenestrationsto reach the target hepatocytes. Alternatively, a lipid nanoparticle maybe sized such that the dimensions of the liposome are of a sufficientdiameter to limit or expressly avoid distribution into certain cells ortissues. For example, lipid nanoparticles that comprise thepharmaceutical and liposomal compositions described herein may be sizedsuch that their dimensions are larger than the fenestrations of theendothelial layer lining hepatic sinusoids to thereby limit distributionof the liposomal lipid nanoparticle to hepatocytes. In such anembodiment, large liposomal compositions (e.g., lipid nanoparticles)will not easily penetrate the endothelial fenestrations, and wouldinstead be cleared by the macrophage Kupffer cells that line the liversinusoids. Sizing of, for example, the lipid nanoparticles comprisingthe pharmaceutical composition may therefore provide an opportunity tofurther manipulate and precisely control the degree to which expressionof the encapsulated polynucleotides may be enhanced in one or moretarget cells. Generally, the size of at least one of the lipidnanoparticles that comprise the pharmaceutical and liposomalcompositions of the present invention is within the range of about 25 to250 nm, preferably less than about 250 nm, 175 nm, 150 nm, 125 nm, 100nm, 75 nm, 50 nm, 25 nm or 10 nm.

Similarly, the compositions of the present invention may be prepared topreferentially distribute to other target tissues, cells or organs, suchas the heart, lungs, kidneys, spleen. For example, the lipidnanoparticles of the present invention may be prepared to achieveenhanced delivery to the target cells and tissues. Accordingly, thecompositions of the present invention may be enriched with additionalcationic, non-cationic and PEG-modified lipids to further target tissuesor cells.

In some embodiments, the compounds and the pharmaceutical and liposomalcompositions described herein (e.g., HGT5002-based lipid nanoparticles)distribute to the cells and tissues of the liver to enhance thedelivery, transfection and the subsequent expression of thepolynucleotides (e.g., mRNA) encapsulated therein by the cells andtissues of the liver (e.g., hepatocytes) and the correspondingproduction of the polypeptide or protein encoded by such polynucleotide.While such compositions may preferentially distribute into the cells andtissues of the liver, the therapeutic effects of the expressedpolynucleotides and the subsequent production of a protein encodedthereby need not be limited to the target cells and tissues. Forexample, the targeted cells (e.g., hepatocytes) may function as a“reservoir” or “depot” capable of expressing or producing, andsystemically or peripherally excreting a functional protein or enzyme,as disclosed for example, in International Application No.PCT/US2010/058457 and in U.S. Provisional Application No. 61/494,881,the teachings of which are both incorporated herein by reference intheir entirety. Accordingly, in certain embodiments of the presentinvention the one or more of the lipid nanoparticles that comprise thepharmaceutical and liposomal compositions described herein (e.g.,HGT5000-based lipid nanoparticles) may target hepatocytes and/orpreferentially distribute to the cells and tissues of the liver upondelivery. Following the transfection of the target hepatocytes by thepolynucleotide encapsulated in one or more of such lipid nanoparticles,such polynucleotides are expressed (e.g., translated) and a functionalproduct (e.g., a polypeptide or protein) is excreted and systemicallydistributed, where such functional product may exert a desiredtherapeutic effect.

The polynucleotides encapsulated in one or more of the compounds orpharmaceutical and liposomal compositions described herein can bedelivered to and/or transfect targeted cells or tissues. In someembodiments, the encapsulated polynucleotides are capable of beingexpressed and functional polypeptide products produced (and in someinstances excreted) by the target cell, thereby conferring a beneficialproperty to, for example the target cells or tissues. Such encapsulatedpolynucleotides may encode, for example, a hormone, enzyme, receptor,polypeptide, peptide or other protein of interest. In certainembodiments, such encapsulated polynucleotides may also encode a smallinterfering RNA (siRNA) or antisense RNA for the purpose of modulatingor otherwise decreasing or eliminating the expression of an endogenousnucleic acid or gene. In certain embodiments such encapsulatedpolynucleotides may be natural or recombinant in nature and may exerttheir therapeutic activity using either sense or antisense mechanisms ofaction (e.g., by modulating the expression of a target gene or nucleicacid).

Also contemplated by the present invention is the co-delivery of one ormore unique polynucleotides to target cells by the compounds orpharmaceutical and liposomal compositions described herein, for example,by combining two unique therapeutic agents or polynucleotides into asingle lipid nanoparticle. Also contemplated is the delivery of one ormore encapsulated polynucleotides to one or more target cells to treat asingle disorder or deficiency, wherein each such polynucleotidefunctions by a different mechanism of action. For example, thepharmaceutical or liposomal compositions of the present invention maycomprise a first polynucleotide which, for example, is encapsulated in alipid nanoparticle and intended to correct an endogenous protein orenzyme deficiency, and a second polynucleotide intended to deactivate or“knock-down” a malfunctioning endogenous polynucleotide and its proteinor enzyme product. Such encapsulated polynucleotides may encode, forexample mRNA and siRNA.

While in vitro transcribed polynucleotides (e.g., mRNA) may betransfected into target cells, such polynucleotides may be readily andefficiently degraded by the cell in vivo, thus rendering suchpolynucleotides ineffective. Moreover, some polynucleotides are unstablein bodily fluids (particularly human serum) and can be degraded ordigested even before reaching a target cell. In addition, within a cell,a natural mRNA can decay with a half-life of between 30 minutes andseveral days. Accordingly, in certain embodiments, the encapsulatedpolynucleotides provided herein, and in particular the mRNApolynucleotides provided herein, preferably retain at least some abilityto be expressed or translated, to thereby produce a functional proteinor enzyme within one or more target cells.

In certain embodiments, the pharmaceutical and liposomal compositionscomprise one or more of the lipid compounds disclosed herein and one ormore lipid nanoparticles that include or encapsulate one or morestabilized polynucleotides (e.g., mRNA which has been stabilized againstin vivo nuclease digestion or degradation) that modulate the expressionof a gene or that may be expressed or translated to produce a functionalpolypeptide or protein within one or more target cells. In certainembodiments, the activity of such encapsulated polynucleotides (e.g.,mRNA encoding a functional protein or enzyme) is prolonged over anextended period of time. For example, the activity of thepolynucleotides may be prolonged such that the pharmaceuticalcompositions may be administered to a subject on a semi-weekly orbi-weekly basis, or more preferably on a monthly, bi-monthly, quarterlyor an annual basis. The extended or prolonged activity of thepharmaceutical compositions of the present invention, and in particularof the encapsulated mRNA, is directly related to the quantity offunctional protein or enzyme translated from such mRNA. Similarly, theactivity of the compositions of the present invention may be furtherextended or prolonged by chemical modifications made to further improveor enhance translation of the mRNA polynucleotides. For example, theKozac consensus sequence plays a role in the initiation of proteintranslation, and the inclusion of such a Kozac consensus sequence in theencapsulated mRNA polynucleotides may further extend or prolong theactivity of the mRNA polynucleotides. Furthermore, the quantity offunctional protein or enzyme produced by the target cell is a functionof the quantity of polynucleotide (e.g., mRNA) delivered to the targetcells and the stability of such polynucleotide. To the extent that thestability of the polynucleotides encapsulated by the compounds orcompositions of the present invention may be improved or enhanced, thehalf-life, the activity of the translated protein or enzyme and thedosing frequency of the composition may be further extended.

In certain embodiments the polynucleotides can be chemically modifiedfor example, to confer stability (e.g., stability relative to thewild-type or naturally-occurring version of the mRNA and/or the versionof the mRNA naturally endogenous to target cells). Accordingly, in someembodiments, the encapsulated polynucleotides provided herein compriseat least one chemical modification which confers increased or enhancedstability to the polynucleotide, including, for example, improvedresistance to nuclease digestion in vivo. The terms “stable” and“stability” as such terms relate to the polynucleotides encapsulated bythe compounds or pharmaceutical and liposomal compositions of thepresent invention, and particularly with respect to the mRNA, refer toincreased or enhanced resistance to degradation by, for examplenucleases (i.e., endonucleases or exonucleases) which are normallycapable of degrading such RNA. Increased stability can include, forexample, less sensitivity to hydrolysis or other destruction byendogenous enzymes (e.g., endonucleases or exonucleases) or conditionswithin the target cell or tissue, thereby increasing or enhancing theresidence of such polynucleotides in the target cell, tissue, subjectand/or cytoplasm. The stabilized polynucleotide molecules providedherein demonstrate longer half-lives relative to their naturallyoccurring, unmodified counterparts (e.g. the wild-type version of thepolynucleotide).

In certain embodiments, a polynucleotide can be modified by theincorporation 3′ and/or 5′ untranslated (UTR) sequences which are notnaturally found in the wild-type polynucleotide. Also contemplated bythe phrases “chemical modification” and “chemically modified” as suchterms related to the polynucleotides encapsulated by the compounds orpharmaceutical and liposomal compositions of the present invention arealterations which improve or enhance translation of mRNApolynucleotides, including for example, the inclusion of sequences whichfunction in the initiation of protein translation (e.g., the Kozacconsensus sequence). (Kozak, M., Nucleic Acids Res 15 (20): 8125-48(1987)). Chemical modifications also include modifications whichintroduce chemistries which differ from those seen in naturallyoccurring polynucleotides, for example, covalent modifications such asthe introduction of modified nucleotides, (e.g., nucleotide analogs, orthe inclusion of pendant groups which are not naturally found in suchpolynucleotide molecules). In some embodiments, the polynucleotides haveundergone a chemical or biological modification to render them morestable prior to encapsulation in one or more lipid nanoparticles. Incertain embodiments, exemplary chemical modifications that may beintroduced into the polynucleotide include pseudouridine, 2-thiouracil,5-methyl cytidine, 5-methylcytosine, isocytosine, pseudoisocytosine,5-bromouracil, 5-propynyluracil, 6-aminopurine, 2-aminopurine, inosine,diaminopurine and 2-chloro-6-aminopurine cytosine. Exemplary chemicalmodifications to a polynucleotide include the depletion of a base (e.g.,by deletion or by the substitution of one nucleotide for another) orchemical modification of a base.

In addition, suitable modifications include alterations in one or morenucleotides of a codon such that the codon encodes the same amino acidbut is more stable than the codon found in the wild-type version of thepolynucleotide. For example, an inverse relationship between thestability of RNA and a higher number cytidines (C's) and/or uridines(U's) residues has been demonstrated, and RNA devoid of C and U residueshave been found to be stable to most RNases (Heidenreich, et al. J BiolChem 269, 2131-8 (1994)). In some embodiments, the number of C and/or Uresidues in an mRNA sequence is reduced. In a another embodiment, thenumber of C and/or U residues is reduced by substitution of one codonencoding a particular amino acid for another codon encoding the same ora related amino acid. Contemplated modifications to the mRNApolynucleotides encapsulated by the compounds or pharmaceutical andliposomal compositions of the present invention also include theincorporation of pseudouridines. The incorporation of pseudouridinesinto the mRNA polynucleotides encapsulated by the compounds orpharmaceutical and liposomal compositions of the present invention mayenhance stability and translational capacity, as well as diminishingimmunogenicity in vivo. (See, e.g., Karikó, K., et al., MolecularTherapy 16 (11): 1833-1840 (2008)). Substitutions and modifications tothe polynucleotides encapsulated by the compounds or pharmaceutical andliposomal compositions of the present invention may be performed bymethods readily known to one or ordinary skill in the art.

The constraints on reducing the number of C and U residues in a sequencewill likely be greater within the coding region of an mRNA, compared toan untranslated region, (i.e., it will likely not be possible toeliminate all of the C and U residues present in the message while stillretaining the ability of the message to encode the desired amino acidsequence). The degeneracy of the genetic code, however presents anopportunity to allow the number of C and/or U residues that are presentin the sequence to be reduced, while maintaining the same codingcapacity (i.e., depending on which amino acid is encoded by a codon,several different possibilities for modification of RNA sequences may bepossible). For example, the codons for Gly can be altered to GGA or GGGinstead of GGU or GGC.

The term chemical modification also includes, for example, theincorporation of non-nucleotide linkages or modified nucleotides intothe polynucleotide sequences of the present invention (e.g.,end-blocking modifications to one or both the 3′ and 5′ ends of an mRNAmolecule encoding a functional protein or enzyme). Such modificationsmay include the addition of bases to a polynucleotide sequence (e.g.,the inclusion of a poly A tail or a longer poly A tail), the alterationof the 3′ UTR or the 5′ UTR, complexing the polynucleotide with an agent(e.g., a protein or a complementary polynucleotide molecule), andinclusion of elements which change the structure of a polynucleotidemolecule (e.g., which form secondary structures).

The poly A tail is thought to stabilize natural messengers and syntheticsense RNA. Therefore, in certain embodiments a long poly A tail can beadded to an mRNA molecule thus rendering the RNA more stable. Poly Atails can be added using a variety of art-recognized techniques. Forexample, long poly A tails can be added to synthetic or in vitrotranscribed RNA using poly A polymerase (Yokoe, et al. NatureBiotechnology. 1996; 14: 1252-1256). A transcription vector can alsoencode long poly A tails. In addition, poly A tails can be added bytranscription directly from PCR products. Poly A may also be ligated tothe 3′ end of a sense RNA with RNA ligase (see, e.g., Molecular CloningA Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis(Cold Spring Harbor Laboratory Press: 1991 edition)). In certainembodiments, the length of the poly A tail is at least about 90, 200,300, 400 at least 500 nucleotides. In certain embodiments, the length ofthe poly A tail is adjusted to control the stability of a modified sensemRNA molecule of the invention and, thus, the transcription of protein.For example, since the length of the poly A tail can influence thehalf-life of a sense mRNA molecule, the length of the poly A tail can beadjusted to modify the level of resistance of the mRNA to nucleases andthereby control the time course of polynucleotide expression and proteinproduction in a target cell. In certain embodiments, the stabilizedpolynucleotide molecules are sufficiently resistant to in vivodegradation (e.g., by nucleases), such that they may be delivered to thetarget cell without a lipid nanoparticle.

In some embodiments, the encapsulated polynucleotides (e.g., mRNAencoding a deficient protein) may optionally include chemical orbiological modifications which, for example, improve the stabilityand/or half-life of such polynucleotide or which improve or otherwisefacilitate translation of such polynucleotide.

In certain embodiments, the chemical modifications are end-blockingmodification of the one or more polynucleotides which comprise thepharmaceutical compositions of the invention. For example, suchpolynucleotides can be modified by the incorporation 3′ and/or 5′untranslated (UTR) sequences which are not naturally found in thewild-type polynucleotide. In certain embodiments, 3′ and/or 5′ flankingsequence which naturally flanks an mRNA and encodes a second, unrelatedprotein can be incorporated into the nucleotide sequence of an mRNAmolecule encoding a or functional protein in order to modify it. Forexample, 3′ or 5′ sequences from mRNA molecules which are stable (e.g.,globin, actin, GAPDH, tubulin, histone, or citric acid cycle enzymes)can be incorporated into the 3′ and/or 5′ region of a sense mRNApolynucleotide molecule to increase the stability of the sense mRNAmolecule.

Also contemplated by the present invention are modifications to thepolynucleotide sequences made to one or both of the 3′ and 5′ ends ofthe polynucleotide. For example, the present invention contemplatesmodifications to the 5′ end of the polynucleotides (e.g., mRNA) toinclude a partial sequence of a CMV immediate-early 1 (IE1) gene, or afragment thereof to improve the nuclease resistance and/or improve thehalf-life of the polynucleotide. In addition to increasing the stabilityof the mRNA polynucleotide sequence, it has been surprisingly discoveredthe inclusion of a partial sequence of a CMV immediate-early 1 (IE1)gene (e.g., to one or more of the 5′ untranslated region and 3′untranslated region of the mRNA) further enhances the translation of themRNA. Also contemplated is the inclusion of a sequence encoding humangrowth hormone (hGH), or a fragment thereof to one or both of the 3′ and5′ ends of the polynucleotide (e.g., mRNA) to further stabilize thepolynucleotide. Generally, the contemplated chemical modificationsimprove the stability and/or pharmacokinetic properties (e.g.,half-life) of the polynucleotide relative to their unmodifiedcounterparts, and include, for example modifications made to improvesuch polynucleotides' resistance to in vivo nuclease digestion.

In some embodiments, the pharmaceutical composition, the two or morelipid nanoparticles comprised therein or the polynucleotidesencapsulated by such lipid nanoparticles can comprise a stabilizingreagent. The compositions can include one or more formulation reagentsthat bind directly or indirectly to, and stabilize the polynucleotide,thereby enhancing residence time in the cytoplasm of a target cell. Suchreagents preferably lead to an improved half-life of a polynucleotide inthe target cells. For example, the stability of an mRNA and efficiencyof translation may be increased by the incorporation of “stabilizingreagents” that form complexes with the polynucleotides (e.g., mRNA) thatnaturally occur within a cell (see e.g., U.S. Pat. No. 5,677,124).Incorporation of a stabilizing reagent can be accomplished for example,by combining the poly A and a protein with the mRNA to be stabilized invitro before loading or encapsulating the mRNA within the one or morelipid nanoparticles that comprise the pharmaceutical composition.Exemplary stabilizing reagents include one or more proteins, peptides,aptamers, translational accessory protein, mRNA binding proteins, and/ortranslation initiation factors.

Stabilization of the pharmaceutical and liposomal compositions describedherein (e.g., lipid nanoparticles) may also be improved by the use ofopsonization-inhibiting moieties, which are typically large hydrophilicpolymers that are chemically or physically bound or otherwiseincorporated into the lipid nanoparticle (e.g., by the intercalation ofa lipid-soluble anchor into the membrane itself, or by binding directlyto active groups of membrane lipids). These opsonization-inhibitinghydrophilic polymers form a protective surface layer which significantlydecreases the uptake of the liposomes by the macrophage-monocyte systemand reticulo-endothelial system (e.g., as described in U.S. Pat. No.4,920,016, the entire disclosure of which is herein incorporated byreference). For example, delays in the uptake of lipid nanoparticles bythe reticuloendothelial system may be facilitated by the addition of ahydrophilic polymer surface coating onto or into lipid nanoparticles tomask the recognition and uptake of the liposomal-based lipidnanoparticle by the reticuloendothelial system. For example, in certainembodiments, one or more of the lipid nanoparticles that comprise thepharmaceutical compositions disclosed herein comprise apolyethyleneglycol (PEG) polymer or a PEG-modified lipid to furtherenhance delivery of such lipid nanoparticles to the target cell andtissues.

When RNA is hybridized to a complementary polynucleotide molecule (e.g.,DNA or RNA) it may be protected from nucleases. (Krieg, et al. Melton.Methods in Enzymology. 1987; 155, 397-415). The stability of hybridizedmRNA is likely due to the inherent single strand specificity of mostRNases. In some embodiments, the stabilizing reagent selected to complexa polynucleotide is a eukaryotic protein, (e.g., a mammalian protein).In yet another embodiment, the polynucleotide (e.g., mRNA) for use insense therapy can be modified by hybridization to a secondpolynucleotide molecule. If an entire mRNA molecule were hybridized to acomplementary polynucleotide molecule translation initiation may bereduced. In some embodiments the 5′ untranslated region and the AUGstart region of the mRNA molecule may optionally be left unhybridized.Following translation initiation, the unwinding activity of the ribosomecomplex can function even on high affinity duplexes so that translationcan proceed. (Liebhaber. J. Mol. Biol. 1992; 226: 2-13; Monia, et al. JBiol Chem. 1993; 268: 14514-22.) It will be understood that any of theabove described methods for enhancing the stability of polynucleotidesmay be used either alone or in combination with one or more of any ofthe other above-described methods and/or compositions.

In certain embodiments, the pharmaceutical compositions of the presentinvention enhance the delivery of lipid nanoparticle-encapsulatedpolynucleotides to one or more target cells, tissues or organs. In someembodiments, enhanced delivery to one or more target cells comprisesincreasing the amount of polynucleotide that comes in contact or isotherwise delivered to the target cells. In some embodiments, enhancingdelivery to target cells comprises reducing the amount of polynucleotidethat comes into contact with non-target cells. In some embodiments,enhancing delivery to target cells comprises allowing the transfectionof at least some target cells with the encapsulated polynucleotide. Insome embodiments, the level of expression of the polynucleotideencapsulated by the lipid nanoparticles which comprise the subjectpharmaceutical compositions and the corresponding production of thefunctional protein or enzyme encoded thereby is increased in the targetcells.

The polynucleotides encapsulated by the compounds or pharmaceutical andliposomal compositions of the present invention may be optionallycombined with a reporter gene (e.g., upstream or downstream of thecoding region of the polynucleotide) which, for example, facilitates thedetermination of polynucleotide delivery to the target cells or tissues.Suitable reporter genes may include, for example, Green FluorescentProtein mRNA (GFP mRNA), Renilla Luciferase mRNA (Luciferase mRNA),Firefly Luciferase mRNA, or any combinations thereof. For example, GFPmRNA may be fused with a polynucleotide encoding GLA mRNA (SEQ ID NO: 4)or EPO mRNA (SEQ ID NO: 1) to facilitate confirmation of mRNAlocalization in the plasma or in one or more target cells, tissues ororgans.

In some embodiments, the pharmaceutical compositions of the presentinvention comprise one or more additional molecules (e.g., proteins,peptides, aptamers or oliogonucleotides) which facilitate the transferof the polynucleotides (e.g., mRNA, miRNA, snRNA and snoRNA) from thelipid nanoparticle into an intracellular compartment of the target cell.In some embodiments, the additional molecule facilitates the delivery ofthe polynucleotides into, for example, the cytosol, the lysosome, themitochondrion, the nucleus, the nucleolae or the proteasome of a targetcell. Also included are agents that facilitate the transport of thetranslated protein of interest from the cytoplasm to its normalintercellular location (e.g., in the mitochondrion) to treatdeficiencies in that organelle. In some embodiments, the agent isselected from the group consisting of a protein, a peptide, an aptamer,and an oligonucleotide.

In some embodiments, the compositions of the present inventionfacilitate a subject's endogenous production of one or more functionalproteins and/or enzymes, and in particular the production of proteinsand/or enzymes which demonstrate less immunogenicity relative to theirrecombinantly-prepared counterparts. In a certain embodiments of thepresent invention, the lipid nanoparticles comprise polynucleotideswhich encode mRNA of a deficient protein or enzyme. Upon distribution ofsuch compositions to the target tissues and the subsequent transfectionof such target cells, the exogenous mRNA loaded or encapsulated into thelipid nanoparticles that comprise the compositions may be translated invivo to produce a functional protein or enzyme encoded by suchencapsulated mRNA (e.g., a protein or enzyme in which the subject isdeficient). Accordingly, in certain embodiments the compositions of thepresent invention exploit a subject's ability to translate exogenously-or recombinantly-prepared mRNA to produce an endogenously-translatedprotein or enzyme, and thereby produce (and where applicable excrete) afunctional protein or enzyme. The translated proteins or enzymes mayalso be characterized by the in vivo inclusion of nativepost-translational modifications which may often be absent inrecombinantly-prepared proteins or enzymes, thereby further reducing theimmunogenicity of the translated protein or enzyme.

The encapsulation of mRNA in the lipid nanoparticles and theadministration of the pharmaceutical compositions comprising such lipidnanoparticles avoid the need to deliver the mRNA to specific organelleswithin a target cell (e.g., mitochondria). Rather, upon transfection ofa target cell and delivery of the encapsulated mRNA to the cytoplasm ofthe target cell, the mRNA contents of the lipid nanoparticles may betranslated and a functional protein or enzyme produced.

The present invention also contemplates the discriminatory targeting ofone or more target cells and tissues by both passive and activetargeting means. The phenomenon of passive targeting exploits thenatural distributions patterns of lipid nanoparticles in vivo withoutrelying upon the use of additional excipients or means to enhancerecognition of the lipid nanoparticle by one or more target cells. Forexample, lipid nanoparticles which are subject to phagocytosis by thecells of the reticulo-endothelial system are likely to accumulate in theliver or spleen, and accordingly may provide means to passively directthe delivery of the compositions to such target cells.

Alternatively, the present invention contemplates active targeting,which involves the use of additional excipients, referred to herein as“targeting ligands” that may be bound (either covalently ornon-covalently) to the lipid nanoparticle to encourage localization ofsuch lipid nanoparticle at certain target cells or target tissues. Forexample, targeting may be mediated by the inclusion of one or moreendogenous targeting ligands (e.g., apolipoprotein E) in or on the lipidnanoparticle to encourage distribution to the target cells or tissues.Recognition of the targeting ligand by the target tissues activelyfacilitates tissue distribution to, and cellular uptake of the lipidnanoparticles and/or their contents by the target cells and tissues. Forexample, in certain embodiments, one or more of the lipid nanoparticlesthat comprise the pharmaceutical formulation may comprise anapolipoprotein-E targeting ligand in or on such lipid nanoparticles tofacilitate or encourage recognition and binding of such lipidnanoparticle to endogenous low density lipoprotein receptors expressed,for example by hepatocytes. As provided herein, the composition cancomprise a ligand capable of enhancing affinity of the compositions toone or more target cells. Targeting ligands may be linked to the outerbilayer of the lipid nanoparticle during formulation orpost-formulation. These methods are well known in the art. In addition,some lipid nanoparticles may comprise fusogenic polymers such as PEAA,hemagluttinin, other lipopeptides (see U.S. patent application Ser. Nos.08/835,281, and 60/083,294, which are incorporated herein by reference)and other features useful for in vivo and/or intracellular delivery. Inother embodiments, the compositions of the present invention demonstrateimproved transfection efficacies, and/or demonstrate enhancedselectivity towards target cells or tissues of interest. Contemplatedtherefore are compositions or lipid nanoparticles that comprise one ormore ligands (e.g., peptides, aptamers, oligonucleotides, a vitamin orother molecules) that are capable of enhancing the affinity of thecompositions or their constituent lipid nanoparticles and theirpolynucleotide contents to one or more target cells or tissues. Suitableligands may optionally be bound or linked to the surface of the lipidnanoparticle. In some embodiments, the targeting ligand may span thesurface of a lipid nanoparticle or be encapsulated within the lipidnanoparticle. Suitable ligands are selected based upon their physical,chemical or biological properties (e.g., selective affinity and/orrecognition of target cell surface markers or features.) Cell-specifictarget sites and their corresponding targeting ligand can vary widely.Suitable targeting ligands are selected such that the uniquecharacteristics of a target cell are exploited, thus allowing thecomposition to discriminate between target and non-target cells. Forexample, compositions of the present invention may bear surface markers(e.g., apolipoprotein-B or apolipoprotein-E) that selectively enhancerecognition of, or affinity to hepatocytes (e.g., by receptor-mediatedrecognition of and binding to such surface markers). Additionally, theuse of galactose as a targeting ligand would be expected to direct thecompositions of the present invention to parenchymal hepatocytes, oralternatively the use of mannose containing sugar residues as atargeting ligand would be expected to direct the compositions of thepresent invention to liver endothelial cells (e.g., mannose containingsugar residues that may bind preferentially to the asialoglycoproteinreceptor present in hepatocytes). (See Hillery A M, et al. “DrugDelivery and Targeting: For Pharmacists and Pharmaceutical Scientists”(2002) Taylor & Francis, Inc.) The presentation of such targetingligands that have been conjugated to moieties present in the lipidnanoparticle therefore facilitate recognition and uptake of theliposomal compositions of the present invention by one or more targetcells and tissues. Examples of suitable targeting ligands include one ormore peptides, proteins, aptamers, vitamins and oligonucleotides.

As used herein, the term “subject” refers to any animal (e.g., amammal), including, but not limited to, humans, non-human primates,rodents, and the like, to which the compounds, pharmaceutical orliposomal compositions and methods of the present invention may beadministered. Typically, the terms “subject” and “patient” are usedinterchangeably herein in reference to a human subject.

The ability of the compounds and pharmaceutical or liposomalcompositions described herein (e.g., lipid nanoparticles) to modulate orenhance the expression of encapsulated polynucleotides and theproduction of a polypeptide or protein provides novel and more efficientmeans of effectuating the in vivo production of polypeptides andproteins for the treatment of a host of diseases or pathologicalconditions. Such lipid nanoparticle compositions are particularlysuitable for the treatment of diseases or pathological conditionsassociated with the aberrant expression of nucleic acids encoding aprotein or enzyme. For example, the successful delivery ofpolynucleotides such as mRNA to target organs such as the liver and inparticular, to hepatocytes, can be used for the treatment and thecorrection of in-born errors of metabolism that are localized to theliver. Accordingly, the compounds, pharmaceutical compositions andrelated methods described herein may be employed to treat a wide rangeof diseases and pathological conditions, in particular those diseaseswhich are due to protein or enzyme deficiencies. The polynucleotidesencapsulated by the compounds or pharmaceutical and liposomalcompositions described herein (e.g., HGT5001-based lipid nanoparticles)may encode a functional product (e.g., a protein, enzyme, polypeptide,peptide, functional RNA, and/or antisense molecule), and preferablyencodes a product whose in vivo production is desired.

The compounds, pharmaceutical compositions and related methods of thepresent invention are broadly applicable to the delivery of therapeuticagents such as polynucleotides, and in particular mRNA, to treat anumber of disorders. In particular, such compounds, compositions andrelated methods of the present invention are suitable for the treatmentof diseases or disorders relating to the deficiency of proteins and/orenzymes. In certain embodiments, the lipid nanoparticle-encapsulatedpolynucleotides encode functional proteins or enzymes that are excretedor secreted by one or more target cells into the surroundingextracellular fluid (e.g., mRNA encoding hormones andneurotransmitters). Alternatively, in another embodiment, thepolynucleotides encapsulated by the compounds or pharmaceutical andliposomal compositions of the present invention encode functionalproteins or enzymes that remain in the cytosol of one or more targetcells (e.g., mRNA encoding an enzyme associated with urea cycle orlysosomal storage metabolic disorders). Other disorders for which thecompounds, pharmaceutical compositions and related methods of thepresent invention are useful include, but are not limited to, disorderssuch as SMN1-related spinal muscular atrophy (SMA); amyotrophic lateralsclerosis (ALS); GALT-related galactosemia; Cystic Fibrosis (CF);SLC3A1-related disorders including cystinuria; COL4A5-related disordersincluding Alport syndrome; galactocerebrosidase deficiencies; X-linkedadrenoleukodystrophy and adrenomyeloneuropathy; Friedreich's ataxia;Pelizaeus-Merzbacher disease; TSC1 and TSC2-related tuberous sclerosis;Sanfilippo B syndrome (MPS IIIB); CTNS-related cystinosis; theFMR1-related disorders which include Fragile X syndrome, FragileX-Associated Tremor/Ataxia Syndrome and Fragile X Premature OvarianFailure Syndrome; Prader-Willi syndrome; Fabry disease; hereditaryhemorrhagic telangiectasia (AT); Niemann-Pick disease Type C1; theneuronal ceroid lipofuscinoses-related diseases including JuvenileNeuronal Ceroid Lipofuscinosis (JNCL), Juvenile Batten disease,Santavuori-Haltia disease, Jansky-Bielschowsky disease, and PTT-1 andTPP1 deficiencies; EIF2B1, EIF2B2, EIF2B3, EIF2B4 and EIF2B5-relatedchildhood ataxia with central nervous system hypomyelination/vanishingwhite matter; CACNA1A and CACNB4-related Episodic Ataxia Type 2; theMECP2-related disorders including Classic Rett Syndrome, MECP2-relatedSevere Neonatal Encephalopathy and PPM-X Syndrome; CDKL5-relatedAtypical Rett Syndrome; Kennedy's disease (SBMA); Notch-3 relatedcerebral autosomal dominant arteriopathy with subcortical infarcts andleukoencephalopathy (CADASIL); SCN1A and SCN1B-related seizuredisorders; the Polymerase G-related disorders which includeAlpers-Huttenlocher syndrome, POLG-related sensory ataxic neuropathy,dysarthria, and ophthalmoparesis, and autosomal dominant and recessiveprogressive external ophthalmoplegia with mitochondrial DNA deletions;X-Linked adrenal hypoplasia; X-linked agammaglobulinemia; and Wilson'sdisease. In certain embodiments, the polynucleotides, and in particularmRNA, of the present invention may encode functional proteins orenzymes. For example, the compositions of the present invention mayinclude mRNA encoding ornithine transcarbamylase (OTC),carbamoyl-phosphate synthetase 1 (CPS1), argininosuccinate synthetase(ASS1), argininosuccinate lyase (ASL) or arginase 1 (ARG1), cysticfibrosis transmembrane conductance regulator (CFTR), acid alphaglucosidase, arylsulfatase A, α-galactosidase A, erythropoietin (e.g.,SED ID NO: 4), α1-antitrypsin, carboxypeptidase N, alpha-L-iduronidase,iduronate-2-sulfatase, iduronate sulfatase,N-acetylglucosamine-1-phosphate transferase, N-acetylglucosaminidase,alpha-glucosaminide acetyltransferase, N-acetylglucosamine 6-sulfatase,N-acetylgalactosamine-4-sulfatase, beta-glucosidase, galactose-6-sulfatesulfatase, beta-galactosidase, beta-glucuronidase, glucocerebrosidase,heparan sulfamidase, heparin-N-sulfatase, lysosomal acid lipase,hyaluronidase, galactocerebrosidase, human growth hormone, survivalmotor neuron, Factor VIII, Factor IX or low density lipoproteinreceptors.

In one embodiment, the mRNA encodes a protein or an enzyme selected fromthe group consisting of human growth hormone, erythropoietin,al-antitrypsin, acid alpha glucosidase, arylsulfatase A,carboxypeptidase N, α-galactosidase A, alpha-L-iduronidase,iduronate-2-sulfatase, iduronate sulfatase,N-acetylglucosamine-1-phosphate transferase, N-acetylglucosaminidase,alpha-glucosaminide acetyltransferase, N-acetylglucosamine 6-sulfatase,N-acetylgalactosamine-4-sulfatase, beta-glucosidase, galactose-6-sulfatesulfatase, beta-galactosidase, beta-glucuronidase, glucocerebrosidase,heparan sulfamidase, heparin-N-sulfatase, lysosomal acid lipase,hyaluronidase, galactocerebrosidase, ornithine transcarbamylase (OTC),carbamoyl-phosphate synthetase 1 (CPS1), argininosuccinate synthetase(ASS1), argininosuccinate lyase (ASL), arginase 1 (ARG1), cysticfibrosis transmembrane conductance regulator (CFTR), survival motorneuron (SMN), Factor VIII, Factor IX and low density lipoproteinreceptors (LDLR).

The compounds and pharmaceutical compositions described herein may beadministered to a subject. In some embodiments, the compositions areformulated in combination with one or more additional polynucleotides,carriers, targeting ligands or stabilizing reagents or other suitableexcipients. Techniques for formulation and administration of drugs maybe found in “Remington's Pharmaceutical Sciences,” Mack Publishing Co.,Easton, Pa., latest edition.

The compounds and the pharmaceutical and liposomal compositions (e.g.,lipid nanoparticles) of the present invention may be administered anddosed in accordance with current medical practice, taking into accountthe clinical condition of the subject, the nature of the encapsulatedmaterials, the site and method of administration, the scheduling ofadministration, the subject's age, sex, body weight and other factorsrelevant to clinicians of ordinary skill in the art. The “effectiveamount” for the purposes herein may be determined by such relevantconsiderations as are known to those of ordinary skill in experimentalclinical research, pharmacological, clinical and medical arts. In someembodiments, the amount administered is effective to achieve at leastsome stabilization, improvement or elimination of symptoms and otherindicators as are selected as appropriate measures of disease progress,regression or improvement by those of skill in the art. For example, asuitable amount and dosing regimen is one that causes at least transientexpression of the one or more polynucleotides in the target cells.

Suitable routes of administration of the compounds and pharmaceuticalcompositions disclosed herein include, for example, oral, rectal,vaginal, transmucosal, or intestinal administration; parenteraldelivery, including intramuscular, subcutaneous, intramedullaryinjections, as well as intrathecal, intracerebroventricular, directintraventricular, intravenous, intraperitoneal, intranasal, orintraocular injections or infusions. In certain embodiments, theadministration of the compounds or compositions (e.g., lipidnanoparticle) described herein to a subject facilitates the contactingof such compounds or compositions to one or more target cells, tissuesor organs.

Alternately, the compounds and compositions of the present invention maybe administered in a local rather than systemic manner, for example, viainjection or infusion of the pharmaceutical compositions directly into atargeted tissue, preferably in a depot or sustained release formulation,such that the contacting of the targeted cells with the constituentlipid nanoparticles may be further facilitated. Local delivery can beaffected in various ways, depending on the tissue to be targeted. Forexample, aerosols containing compositions of the present invention canbe inhaled (for nasal, tracheal, or bronchial delivery); compositions ofthe present invention can be injected into the site of injury, diseasemanifestation, or pain, for example; compositions can be provided inlozenges for oral, tracheal, or esophageal application; can be suppliedin liquid, tablet or capsule form for administration to the stomach orintestines, can be supplied in suppository form for rectal or vaginalapplication; or can even be delivered to the eye by use of creams,drops, or even injection. Formulations containing the compounds of thepresent invention complexed with therapeutic molecules or ligands caneven be surgically administered, for example in association with apolymer or other structure or substance that can allow the compositionsto diffuse from the site of implantation to surrounding cells.Alternatively, such compositions can be applied surgically without theuse of polymers or supports.

Also contemplated herein are lyophilized pharmaceutical compositionscomprising one or more of the compounds disclosed herein and relatedmethods for the use of such lyophilized compositions as disclosed forexample, in U.S. Provisional Application No. 61/494,882, filed Jun. 8,2011, the teachings of which are incorporated herein by reference intheir entirety.

In certain embodiments, the compositions of the present invention areformulated such that they are suitable for extended-release of the, forexample, polynucleotides or nucleic acids encapsulated therein. Suchextended-release compositions may be conveniently administered to asubject at extended dosing intervals. For example, in certainembodiments, the compositions of the present invention are administeredto a subject twice day, daily or every other day. In a certainembodiments, the compositions of the present invention are administeredto a subject twice a week, once a week, every ten days, every two weeks,every three weeks, or more preferably every four weeks, once a month,every six weeks, every eight weeks, every other month, every threemonths, every four months, every six months, every eight months, everynine months or annually. Also contemplated are compositions and lipidnanoparticles which are formulated for depot administration (e.g.,intramuscularly, subcutaneously, intravitreally) to either deliver orrelease a polynucleotide (e.g., mRNA) over extended periods of time.Preferably, the extended-release means employed are combined withmodifications (e.g., chemical modifications) introduced into thepolynucleotides to enhance stability.

While certain compounds, compositions and methods of the presentinvention have been described with specificity in accordance withcertain embodiments, the following examples serve only to illustrate thecompounds of the invention and are not intended to limit the same. Eachof the publications, reference materials and the like referenced hereinto describe the background of the invention and to provide additionaldetail regarding its practice are hereby incorporated by reference intheir entirety.

The articles “a” and “an” as used herein in the specification and in theclaims, unless clearly indicated to the contrary, should be understoodto include the plural referents. Claims or descriptions that include“or” between one or more members of a group are considered satisfied ifone, more than one, or all of the group members are present in, employedin, or otherwise relevant to a given product or process unless indicatedto the contrary or otherwise evident from the context. The inventionincludes embodiments in which exactly one member of the group is presentin, employed in, or otherwise relevant to a given product or process.The invention also includes embodiments in which more than one, or theentire group members are present in, employed in, or otherwise relevantto a given product or process. Furthermore, it is to be understood thatthe invention encompasses all variations, combinations, and permutationsin which one or more limitations, elements, clauses, descriptive terms,etc., from one or more of the listed claims is introduced into anotherclaim dependent on the same base claim (or, as relevant, any otherclaim) unless otherwise indicated or unless it would be evident to oneof ordinary skill in the art that a contradiction or inconsistency wouldarise. Where elements are presented as lists, (e.g., in Markush group orsimilar format) it is to be understood that each subgroup of theelements is also disclosed, and any element(s) can be removed from thegroup. It should be understood that, in general, where the invention, oraspects of the invention, is/are referred to as comprising particularelements, features, etc., certain embodiments of the invention oraspects of the invention consist, or consist essentially of, suchelements, features, etc. For purposes of simplicity those embodimentshave not in every case been specifically set forth in so many wordsherein. It should also be understood that any embodiment or aspect ofthe invention can be explicitly excluded from the claims, regardless ofwhether the specific exclusion is recited in the specification. Thepublications and other reference materials referenced herein to describethe background of the invention and to provide additional detailregarding its practice are hereby incorporated by reference.

EXAMPLES Example 1

The compound(15Z,18Z)—N,N-dimethyl-6-(9Z,12Z)-octadeca-9,12-dien-1-yl)tetracosa-15,18-dien-1-amine(referred to herein as “HGT5000”) was prepared in accordance with thegeneral synthetic scheme shown below in Reaction 1.

The intermediate compound(15Z,18Z)-6-[(9Z,12Z)-octadeca-9,12-dien-1-yl]tetracosa-15,18-diene-1,6-diolidentified as compound (2) in Reaction 1 above was prepared as follows.To a 100 mL round bottom flask was added 10 g (30 mmol) of compound (1)(linoleyl bromide) and dry THF (20 mL) under nitrogen. Magnesium powder(1.11 g, 45 mmol) was added to the stirred reaction solution followed by2 drops of dibromoethane at room temperature. The reaction mixture wasstirred at 50° C. for 1 hour, and then diluted with dry THF (40 mL). Thereaction mixture was stirred another 15 minutes at room temperature.

In a separate 250 mL 3-neck flask was taken ε-caprolactone (1.44 mL,13.5 mmol) in dry THF (20 mL) under nitrogen. To the stirred solutionwas added the Grignard reagent through a cannula at 0° C. The resultingmixture was heated at 85° C. for 3 hours. After cooling to roomtemperature, the reaction mixture was then quenched with NH₄Cl solutionand extracted with dichloromethane (3×100 mL). The combined extractswere washed with brine (50 mL), dried (Na₂SO₄) and concentrated. Theresidue was purified twice by silica gel column chromatography (gradientelution from hexane to 3:2 hexane/EA) to afford compound (2) as an oil.Yield: 5.46 g (65%). ¹H NMR (301 MHz, CDCl₃) δ: 5.25-5.45 (m, 8H), 3.65(m, 2H), 2.77 (t, J=6.2 Hz, 4H), 1.95-2.1 (m, 8H), 1.2-1.70 (m, 50H),0.88 (t, J=6.9 Hz, 6H).

The intermediate compound(15Z,18Z)-6-[(9Z,12Z)-octadeca-9,12-dien-1-yl]tetracosa-15,18-diene-1-olidentified as compound (3) in Reaction 1 above was prepared as follows.Compound (2) (4.4 g, 7.15 mmol) was dissolved in dichloromethane (70ml). The solution was stirred under nitrogen at 0° C. and Et₃SiH (8.07mL, 50.08 mmol) was added. Boron trifluoride diethyl etherate (8.77 mL,71.5 mmol) was added dropwise at 0° C. The reaction mixture was thenstirred at the same temperature for 3 hours, then at room temperaturefor 30 minutes. The reaction was then quenched by 10% sodium carbonatesolution (200 mL). The resulting mixture was extracted twice withdichloromethane (2×150 mL). The combined extract was washed with brine,dried over sodium sulfate, filtered and concentrated under reducedpressure. The crude product was purified twice by silica gel columnchromatography (gradient elution from hexane to 2:1 hexane/EA) to affordthe desired intermediate product compound (3) as an oil. Yield: 3.86 g(90%). ¹H NMR (301 MHz, CDCl₃) δ: 5.2-5.5 (m, 8H), 3.62 (q, J=6.6 Hz,2H), 2.77 (t, J=6 Hz, 4H), 1.9-2.1 (m, 8H), 1.5-1.65 (m, 2H), 1.1-1.45(m, 48H), 0.88 (t, J=6.9 Hz, 6H).

The intermediate compound(6Z,9Z,28Z,31Z)-19-(5-bromopentyl)heptatriaconta-6,9,28,31-tetraeneidentified as compound (4) in Reaction 1 above was prepared as follows.A solution of compound (3) (3.86 g, 6.45 mmol) in dichloromethane (80mL) was stirred under nitrogen at 0° C. Triphenylphosphine (1.86 g, 7.10mmol) was added to the solution followed by tetrabromomethane (2.14 g,6.45 mmol). The reaction mixture was stirred at 0° C. for 3 hours, thenat room temperature for 30 minutes. TLC still showed a presence ofstarting material, accordingly another portion of triphenylphosphine(0.4 g) was added at 0° C. After 30 minutes, all the starting materialhad been consumed and the reaction mixture was then concentrated. To theresidue was added a mixture of ether and hexane (2:1, 200 mL) and theslurry stirred for 15 minutes. Solids were filtered off and the filtratewas concentrated under reduced pressure. The residue was purified bymultiple column chromatographies (gradient elution from hexane to 3:2hexane/EA) to afford the desired intermediate product compound (4).Yield: 2.7 g (63%). ¹H NMR (301 MHz, CDCl₃) δ: 5.2-5.5 (m, 8H), 3.40 (t,J=7 Hz, 2H), 2.77 (t, J=6 Hz, 4H), 1.8-2.1 (m, 8H), 1.15-1.5 (m, 46H),0.88 (t, J=6.6 Hz, 6H).

To prepare the HGT5000 compound(15Z,18Z)—N,N-dimethyl-6-(9Z,12Z)-octadeca-9,12-dien-1-yl)tetracosa-15,18-dien-1-amine),compound (4) (2.70 g, 4.07 mmol) was dissolved in a 2M solution ofdimethylamine in THF (204 mL, 100 eq.). The resulting solution wasstirred under nitrogen at room temperature for 16 hours. The reactionmixture was then concentrated under reduced pressure. The residue waspurified twice by silica gel column chromatography (gradient elutionfrom 0%-10% methanol in dichloromethane) to give the HGT5000 compound asa light yellow oil. Yield: 2.52 g (96%). ¹H NMR (301 MHz, CDCl₃) δ:5.42-5.29 (m, 8H), 2.77 (t, J=6.0 Hz, 4H), 2.28-2.24 (m, 8H), 2.01-2.08(m, 8H), 1.66-1.63 (m, 2H), 1.41-1.20 (m, 48H), 0.88 (t, J=6.9 Hz, 6H).¹³C NMR (CDCl₃) δ: 130.3, 128.0, 59.9, 45.3, 37.5, 33.8, 31.6, 30.3,29.8, 29.7, 29.4, 28.0, 27.5, 27.3, 26.8, 26.7, 25.7, 22.7. APCI [M+H]626.6. R_(f)=0.48 (10% MeOH in DCM).

Example 2

The compound(15Z,18Z)—N,N-dimethyl-6-((9Z,12Z)-octadeca-9,12-dien-1-yl)tetracosa-4,15,18-trien-1-amine(referred to herein as “HGT5001”) was prepared in accordance with thegeneral synthetic scheme illustrated below in Reaction 2.

The intermediate compounds(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl formate (6) and(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-ol respectivelyidentified as compounds (6) and (7) in Reaction 2 above were prepared inan oven dried 3-neck 500 mL flask that was charged with Mg (0.5 g, 20.83mmol, 1.37 eq.) and I₂ (one crystal) under argon. The flask was degassedon a high-vacuum line, then flushed with argon (the process was repeatedfour times) and then stirred at room temperature for approximately 5minutes. Anhydrous ether (22 mL) was added to this flask and the slurrystirred for approximately 10 minutes. Next, 5 g (15.2 mmol, 1 eq.) ofcompound (5) (linoleyl bromide) was added under argon (color change wasobserved after the addition of approximately 4.5 mL of compound (5)) andthe reaction stirred at room temperature. An exothermic reaction wasobserved after stirring for approximately 5 minutes at room temperature.Thus, the mixture was cooled using an ice-water bath for approximately 2minutes, then the ice-bath was removed and the reaction mixture stirredat room temperature for 2 hours, resulting in an ash colored reactionmixture and not all of the Mg was consumed. The mixture was cooled to 0°C. and the HCO₂Et (0.58 mL, 7.17 mmol, 0.47 eq.) was added dropwisedirectly into the solution. After stirring at room temperature for 3hours (product was observed after 1 hour by MS and TLC) the mixture wasdecanted and the Mg turnings washed with ether. The combined washingswere diluted with ether (100 mL), washed with 10% H₂SO₄ (2×50 mL),water, brine and then dried (Na₂SO₄). The solution was filtered,concentrated and the residue purified by silica-gel columnchromatography.

5-7% ether in hexanes eluted the alcohol (compound (7)) from theresidue. Yield: 0.34 g (8%). Compound 7: ¹H NMR (300 MHz, CDCl₃): δ5.38-5.31 (m, 8H), 3.58 (br s, 1H), 2.76 (t, J=6 Hz, 4H), 2.04 (q, J=6.8Hz, 8H), 1.39-1.26 (m, 40H), 0.88 (t, J=6.8 Hz, 6H). APCI[M+H] 527, 511(—H₂O).

2% ether in hexanes eluted the formate (compound (6)) from the residue.Yield: 1.7 g (40%). Compound 6: ¹H NMR (300 MHz, CDCl₃): δ 8.08 (s, 1H),5.42-5.28 (m, 8H), 4.99-4.95 (m, 1H), 2.76 (t, J=6 Hz, 4H), 2.04 (q,J=6.6 Hz, 8H), 1.39-1.26 (m, 40H), 0.88 (t, J=6.6 Hz, 6H). APCI[M+H]557. To obtain compound (7) from compound (6), KOH (powder, 0.76 g, 13.5mmol, 1.4 eq.) was added to a cloudy solution of compound (6) (5.33 g,9.59 mmol, 1 eq.) in EtOH/H₂O (90 mL/16 mL). The reaction mixture wasstirred at room temperature overnight under a N₂ atm. This mixture wasthen concentrated, diluted with ether, washed with 5% aq. HCl (2×100mL), water and dried (Na₂SO₄). The solution was filtered, concentratedand then dried under high vacuum to obtain the compound (7) as acolorless oil. Yield: 4.9 g (96%). ¹H NMR (300 MHz, CDCl₃): δ 5.38-5.31(m, 8H), 3.58 (br s, 1H), 2.76 (t, J=6 Hz, 4H), 2.04 (q, J=6.8 Hz, 8H),1.39-1.26 (m, 40H), 0.88 (t, J=6.8 Hz, 6H). APCI[M+H] 527, 511 (—H₂O).

The intermediate compound(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-one identified ascompound (8) in Reaction 2 above was prepared as follows. To a solutionof compound (7) (4.81 g, 9.09 mmol) in anhydrous CH₂Cl₂ (230 mL) wasadded portionwise Na₂CO₃ (0.49 g, 4.54 mmol) and then PCC (4.9 g, 22.7mmol, 2.5 eq.) over a period of 15 minutes. The black mixture wasstirred at room temperature for 1.5 hours. TLC showed completion ofreaction. The reaction mixture was filtered through a silica-gel pad(200 g) and the pad washed with CH₂Cl₂ (3×400 mL). The filtrate wasconcentrated and dried on a high-vacuum line to obtain the ketonecompound (8) as a colorless oil. Yield: 4.5 g (98%). ¹H NMR (300 MHz,CDCl₃): δ 5.36-5.33 (m, 8H), 2.76 (t, J=5.8 Hz, 4H), 2.37 (t, J=7.4 Hz,4H), 2.04 (q, J=6 Hz, 8H), 1.32-1.27 (m, 36H), 0.88 (t, J=6.8 Hz, 6H).APCI[M+H] 527.

The intermediate compound (6Z,9Z,28Z,31Z)-19-(methoxymethylene)heptatriaconta-6,9,28,31-tetraene identified as compound (9) in Reaction2 above was prepared as follows. A mixture of compound (8) (2.7 g, 5.12mmol, 1 eq.) and (methoxymethyl)triphenyl phosphonium chloride (2.63 g,7.67 mmol, 1.5 eq.) was degassed under high vacuum and flushed withargon (4 times). Anhydrous THF (68 mL) was added followed by 1M KOt-Buin THF (7.67 mL, 7.67 mmol, 1.5 eq.) dropwise by syringe. The resultingred solution was stirred at room temperature overnight. The reactionmixture was diluted with ether, washed with water, brine and dried(Na2SO4). Removal of the solvent and chromatography (1-4% ether inhexanes) of the residue yielded the product compound (9) as a colorlessoil. Yield: 2.7 g (95%). 1H NMR (300 MHz, CDCl3): δ 5.72 (s, 1H),5.36-5.33 (m, 8H), 3.5 (s, 3H), 2.76 (t, J=6 Hz, 4H), 2.05-1.98 (m,10H), 1.85-1.80 (m, 2H), 1.31-1.27 (m, 36H), 0.88 (t, J=6.6 Hz, 6H).APCI[M+H] 555.

The intermediate compound(11Z,14Z)-2-((9Z,12Z)-octadeca-9,12-dien-1-yl)icosa-11,14-dienalidentified as compound (10) in Reaction 2 above was prepared as follows.To a cloudy solution of compound (9) (1.3 g, 2.34 mmol) in a dioxane/H₂O(56 mL/29 mL) solution was added 4M HCl in dioxane (29 mL, 116 mmol, 49eq.) at 0° C. dropwise over 10 minutes. The mixture was allowed to warmto room temperature and then stirred at room temperature for 40 hours(monitored by TLC). The mixture was then diluted with ether, cooled to0° C. and then slowly quenched with aqueous NaHCO₃. The organic layerwas separated, washed with brine, dried (Na₂SO₄), filtered, concentratedand the residue purified by silica-gel column chromatography. 1% etherin hexanes eluted the product compound (10) as a colorless oil. Yield:1.21 g (96%). ¹H NMR (300 MHz, CDCl₃): δ 9.53 (d, J=3.3 Hz, 1H),5.36-5.33 (m, 8H), 2.76 (t, J=5.8 Hz, 4H), 2.23-2.18 (m, 1H), 2.05-1.96(m, 8H), 1.61-1.16 (m, 40H), 0.88 (t, J=7 Hz, 6H). APCI[M+H] 541.

The intermediate compound (4-Dimethylaminobutyl) triphenylphosphoniumbromide (compound (14)) was prepared in accordance with the generalsynthetic scheme shown below is shown in Reaction 3.

The intermediate compound (4-Bromobutyl)triphenylphosphonium bromide(compound (12)) depicted in Reaction 3 above was prepared by placing 10g (46.3 mmol) of 1,4-dibromobutane (compound (11)) and 12.1 g PPh₃ (46.3mmol) in dry toluene (74 mL), and the mixture heated to reflux andboiled overnight. The solid that formed was filtered, washed withtoluene and dried under vacuum to provide the product compound (12) as awhite solid. Yield: 16.1 g (73%). ¹H NMR (300 MHz, CDCl₃): δ 7.84-7.68(m, 15H), 4.03-3.93 (m, 2H), 3.58 (t, J=6 Hz, 2H), 2.36-2.28 (m, 2H),1.89-1.76 (m, 2H). APCI[M+H] 397 (M-Br), 399 (M+2-Br).

The intermediate compound (4-Dimethylaminobutyl) triphenylphosphoniumbromide (compound (14)) was then prepared by adding 3 g (6.28 mmol, 1eq.) of compound (12) portionwise to a solution of 2M dimethylamine inTHF (31.4 mL, 62.8 mmol, 10 eq.) at 0° C. under N₂. The resultingsuspension was allowed to stir at room temperature for 4 hours. CH₃CN(35 mL) was then added to this suspension and it was further stirred atroom temperature overnight. Nitrogen gas was then bubbled into thereaction mixture to remove excess dimethylamine and solvents. Theresulting solid was dried under high vacuum and provided the dry productcompound (13) as a light yellow solid. Yield: 3.16 g (96%). The productcompound (13) was stirred with saturated aqueous NaHCO₃ (110 mL) for 15minutes and lyophilized to produce a light yellow solid. This solid wasstirred with chloroform and filtered. The filtrate was dried over MgSO₄,filtered, concentrated and the residue dried under high vacuum at 45° C.to produce the product compound (14) as a light pink solid. Yield: 2.7 g(97%). ¹H NMR (300 MHz, CDCl₃): δ 7.89-7.75 (m, 9H), 7.71-7.65 (m, 6H),3.93-3.83 (m, 2H), 2.47 (t, J=6.8 Hz, 2H), 2.25 (s, 6H), 1.94-1.87 (m,2H), 1.75-1.62 (m, 2H). APCI[M+H] 362 (M-Br).

HGT5001((15Z,18Z)—N,N-dimethyl-6-((9Z,12Z)-octadeca-9,12-dien-1-yl)tetracosa-4,15,18-trien-1-amine)was then prepared by adding charged intermediate compound (14) (0.58 g,1.32 mmol, 1.5 eq.) to a flame dried RB flask (3-neck, 100 mL) and theflask was then equipped with a magnetic stir bar. This set-up wasdegassed (under high vacuum) and flushed with argon (3 times). AnhydrousTHF (10 mL) was then added to the flask with a syringe. The resultingsuspension was stirred under argon for 5 minutes and then cooled to −78°C. KHMDS (1M in THF, 1.32 mL, 1.32 mmol, 1.5 eq.) was then addeddropwise to the reaction flask and resulted in a yellowish orange cloudysolution. This solution was stirred at −78° C. for 45 minutes. Thecooling bath was removed and the reaction stirred at room temperaturefor 15 minutes to give a reddish orange solution. The mixture was cooledagain to −78° C. and a solution of intermediate compound (10) (0.47 g,0.88 mmol) in dry THF (13 mL) was added through a cannula. The reactionmixture color changed to light yellow. The reaction mixture was stirredat −78° C. for 45 minutes and then the cooling bath was removed,stirring was continued at room temperature for an additional 30 minutes.The mixture was cooled again to −20° C. and then quenched with water (7mL). The reaction mixture was diluted with ether and stirred for 10minutes. The organic layer was separated, washed with brine, dried overMgSO₄, filtered, concentrated and the residue purified by columnchromatography on a silica-gel column. 1.5-2% methanol in chloroformeluted the HGT5001 product as a light yellow oil. Yield: 0.43 g (79%).¹H NMR (300 MHz, C₆D₆): δ 5.52-5.46 (m, 9H), 5.22-5.12 (m, 1H), 2.89 (t,J=5.8 Hz, 4H), 2.43 (br s, 1H), 2.24-2.03 (m, 18H), 1.55-1.37 (m, 2H),1.35-1.22 (m, 40H), 0.88 (t, J=6.8 Hz, 6H). APCI[M+H] 624. Elementalanalysis calculated for C₄₄H₈₁N (theory, found): C (84.67, 84.48); H(13.08, 13.12); N (2.24, 2.19).

Example 3

Lipid nanoparticles comprising HGT5000, DOPE, cholesterol andDMG-PEG2000 and encapsulating human erythropoietin (EPO) mRNA wereformed via standard ethanol injection methods. (Ponsa, et al., Int. J.Pharm. (1993) 95: 51-56.) Ethanolic stock solutions of the lipids wereprepared ahead of time at a concentration of 50 mg/mL and stored at −20°C.

Human erythropoietin (EPO) mRNA was synthesized by in vitrotranscription from a plasmid DNA template encoding the gene, which wasfollowed by the addition of a 5′ cap structure (Cap1) (Fechter, P. etal., J. Gen. Virology (2005) 86: 1239-1249) and a 3′ poly(A) tail ofapproximately 200 nucleotides in length as determined by gelelectrophoresis. The 5′ and 3′ untranslated regions present in the EPOmRNA are represented as X and Y in SEQ ID NO: 1, as indicated below.

Human Erythropoietin mRNA: SEQ ID NO: 1XAUGGGGGUGCACGAAUGUCCUGCCUGGCUGUGGCUUCUCCUGUCCCUGCUGUCGCUCCCUCUGGGCCUCCCAGUCCUGGGCGCCCCACCACGCCUCAUCUGUGACAGCCGAGUCCUGGAGAGGUACCUCUUGGAGGCCAAGGAGGCCGAGAAUAUCACGACGGGCUGUGCUGAACACUGCAGCUUGAAUGAGAAUAUCACUGUCCCAGACACCAAAGUUAAUUUCUAUGCCUGGAAGAGGAUGGAGGUCGGGCAGCAGGCCGUAGAAGUCUGGCAGGGCCUGGCCCUGCUGUCGGAAGCUGUCCUGCGGGGCCAGGCCCUGUUGGUCAACUCUUCCCAGCCGUGGGAGCCCCUGCAGCUGCAUGUGGAUAAAGCCGUCAGUGGCCUUCGCAGCCUCACCACUCUGCUUCGGGCUCUGGGAGCCCAGAAGGAAGCCAUCUCCCCUCCAGAUGCGGCCUCAGCUGCUCCACUCCGAACAAUCACUGCUGACACUUUCCGCAAACUCUUCCGAGUCUACUCCAAUUUCCUCCGGGGAAAGCUGAAGCUGUACACAGGGGAGGCCUGCAGGACAGGGGACAGAUGAY X = GGGAUCCUACC (SEQ ID NO: 2) Y =UUUGAAUU (SEQ ID NO: 3)

The EPO mRNA was stored in water at a final concentration of 1 mg/mL at−80° C. until the time of use. All mRNA concentrations were determinedvia the Ribogreen assay (Invitrogen). Encapsulation of mRNA wascalculated by performing the Ribogreen assay with and without thepresence of 0.1% Triton-X 100. Particle sizes (dynamic light scattering(DLS)) and zeta potentials were determined using a Malvern Zetasizerinstrument in 1×PBS and 1 mM KCl solutions, respectively.

Aliquots of 50 mg/mL ethanolic solutions of the HGT5000, DOPE,cholesterol and DMG-PEG2K were mixed and diluted with ethanol to 3 mLfinal volume. Separately, an aqueous buffered solution (10 mMcitrate/150 mM NaCl, pH 4.5) of EPO mRNA was prepared from a 1 mg/mLstock. The lipid solution was injected rapidly into the aqueous mRNAsolution and shaken to yield a final suspension in 20% ethanol. Theresulting nanoparticle suspension was filtered, diafiltrated with 1×PBS(pH 7.4), concentrated and stored at 2-8° C. Final concentration=1.82mg/mL EPO mRNA (encapsulated). Z_(ave)=105.6 nm (Dv₍₅₀₎=53.7 nm;Dv₍₉₀₎=157 nm).

Example 4

Lipid nanoparticles comprising HGT5000, DOPE, cholesterol andDMG-PEG2000 and encapsulating human alpha-galactosidase (GLA) mRNA wereformed via standard ethanol injection methods. (Ponsa, et al., Int. J.Pharm. (1993) 95: 51-56.) Ethanolic stock solutions of the lipids wereprepared ahead of time at a concentration of 50 mg/mL and stored at −20°C.

Human GLA mRNA was synthesized by in vitro transcription from a plasmidDNA template encoding the gene, which was followed by the addition of a5′ cap structure (Cap1) (Fechter, P. et al., J. Gen. Virology (2005) 86:1239-1249) and a 3′ poly(A) tail of approximately 200 nucleotides inlength as determined by gel electrophoresis. The 5′ and 3′ untranslatedregions present in the GLA mRNA are represented as X and Y in SEQ ID NO:4, as indicated below.

Alpha-galactosidase (GLA) mRNA: SEQ ID NO: 4XAUGCAGCUGAGGAACCCAGAACUACAUCUGGGCUGCGCGCUUGCGCUUCGCUUCCUGGCCCUCGUUUCCUGGGACAUCCCUGGGGCUAGAGCACUGGACAAUGGAUUGGCAAGGACGCCUACCAUGGGCUGGCUGCACUGGGAGCGCUUCAUGUGCAACCUUGACUGCCAGGAAGAGCCAGAUUCCUGCAUCAGUGAGAAGCUCUUCAUGGAGAUGGCAGAGCUCAUGGUCUCAGAAGGCUGGAAGGAUGCAGGUUAUGAGUACCUCUGCAUUGAUGACUGUUGGAUGGCUCCCCAAAGAGAUUCAGAAGGCAGACUUCAGGCAGACCCUCAGCGCUUUCCUCAUGGGAUUCGCCAGCUAGCUAAUUAUGUUCACAGCAAAGGACUGAAGCUAGGGAUUUAUGCAGAUGUUGGAAAUAAAACCUGCGCAGGCUUCCCUGGGAGUUUUGGAUACUACGACAUUGAUGCCCAGACCUUUGCUGACUGGGGAGUAGAUCUGCUAAAAUUUGAUGGUUGUUACUGUGACAGUUUGGAAAAUUUGGCAGAUGGUUAUAAGCACAUGUCCUUGGCCCUGAAUAGGACUGGCAGAAGCAUUGUGUACUCCUGUGAGUGGCCUCUUUAUAUGUGGCCCUUUCAAAAGCCCAAUUAUACAGAAAUCCGACAGUACUGCAAUCACUGGCGAAAUUUUGCUGACAUUGAUGAUUCCUGGAAAAGUAUAAAGAGUAUCUUGGACUGGACAUCUUUUAACCAGGAGAGAAUUGUUGAUGUUGCUGGACCAGGGGGUUGGAAUGACCCAGAUAUGUUAGUGAUUGGCAACUUUGGCCUCAGCUGGAAUCAGCAAGUAACUCAGAUGGCCCUCUGGGCUAUCAUGGCUGCUCCUUUAUUCAUGUCUAAUGACCUCCGACACAUCAGCCCUCAAGCCAAAGCUCUCCUUCAGGAUAAGGACGUAAUUGCCAUCAAUCAGGACCCCUUGGGCAAGCAAGGGUACCAGCUUAGACAGGGAGACAACUUUGAAGUGUGGGAACGACCUCUCUCAGGCUUAGCCUGGGCUGUAGCUAUGAUAAACCGGCAGGAGAUUGGUGGACCUCGCUCUUAUACCAUCGCAGUUGCUUCCCUGGGUAAAGGAGUGGCCUGUAAUCCUGCCUGCUUCAUCACACAGCUCCUCCCUGUGAAAAGGAAGCUAGGGUUCUAUGAAUGGACUUCAAGGUUAAGAAGUCACAUAAAUCCCACAGGCACUGUUUUGCUUCAGCUAGAAAAUACAAUGCAGAUGUCAUUAAAAGACUUACUUUAAY X =GGGAUCCUACC (SEQ ID NO: 2) Y = UUUGAAUU (SEQ ID NO: 3)

The GLA mRNA was stored in water at a final concentration of 1 mg/mL at−80° C. until the time of use. All mRNA concentrations were determinedvia the Ribogreen assay (Invitrogen). Encapsulation of mRNA wascalculated by performing the Ribogreen assay with and without thepresence of 0.1% Triton-X 100. Particle sizes (dynamic light scattering(DLS)) and zeta potentials were determined using a Malvern Zetasizerinstrument in 1×PBS and 1 mM KCl solutions, respectively.

Aliquots of 50 mg/mL ethanolic solutions of the HGT5000, DOPE,cholesterol and DMG-PEG2K were mixed and diluted with ethanol to 3 mLfinal volume. Separately, an aqueous buffered solution (10 mMcitrate/150 mM NaCl, pH 4.5) of GLA mRNA was prepared from the 1 mg/mLstock solution. The lipid solution was injected rapidly into the aqueousmRNA solution and shaken to yield a final suspension in 20% ethanol. Theresulting nanoparticle suspension was filtered, diafiltrated with 1×PBS(pH 7.4), concentrated and stored at 2-8° C. Final concentration=1.38mg/mL GLA mRNA (encapsulated). Z_(ave)=77.7 nm (Dv₍₅₀₎=62.3 nm;Dv₍₉₀₎=91.7 nm).

Example 5

Lipid nanoparticles comprising HGT5001, DOPE, cholesterol andDMG-PEG2000 and encapsulating human alpha-galactosidase (GLA) mRNA wereformed via standard ethanol injection methods. (Ponsa, et al., Int. J.Pharm. (1993) 95: 51-56.) Ethanolic stock solutions of the lipids wereprepared ahead of time at a concentration of 50 mg/mL and stored at −20°C.

Human alpha-galactosidase (GLA) mRNA was synthesized by in vitrotranscription from a plasmid DNA template encoding the gene, which wasfollowed by the addition of a 5′ cap structure (Cap1) (Fechter, P. etal., J. Gen. Virology (2005) 86: 1239-1249) and a 3′ poly(A) tail ofapproximately 200 nucleotides in length as determined by gelelectrophoresis. The 5′ and 3′ untranslated regions present in the humanalpha-galactosidase (GLA) mRNA are respectively represented as X and Yin SEQ ID NO: 4, as indicated below.

Human alpha-galactosidase (GLA) mRNA: SEQ ID NO: 4XAUGCAGCUGAGGAACCCAGAACUACAUCUGGGCUGCGCGCUUGCGCUUCGCUUCCUGGCCCUCGUUUCCUGGGACAUCCCUGGGGCUAGAGCACUGGACAAUGGAUUGGCAAGGACGCCUACCAUGGGCUGGCUGCACUGGGAGCGCUUCAUGUGCAACCUUGACUGCCAGGAAGAGCCAGAUUCCUGCAUCAGUGAGAAGCUCUUCAUGGAGAUGGCAGAGCUCAUGGUCUCAGAAGGCUGGAAGGAUGCAGGUUAUGAGUACCUCUGCAUUGAUGACUGUUGGAUGGCUCCCCAAAGAGAUUCAGAAGGCAGACUUCAGGCAGACCCUCAGCGCUUUCCUCAUGGGAUUCGCCAGCUAGCUAAUUAUGUUCACAGCAAAGGACUGAAGCUAGGGAUUUAUGCAGAUGUUGGAAAUAAAACCUGCGCAGGCUUCCCUGGGAGUUUUGGAUACUACGACAUUGAUGCCCAGACCUUUGCUGACUGGGGAGUAGAUCUGCUAAAAUUUGAUGGUUGUUACUGUGACAGUUUGGAAAAUUUGGCAGAUGGUUAUAAGCACAUGUCCUUGGCCCUGAAUAGGACUGGCAGAAGCAUUGUGUACUCCUGUGAGUGGCCUCUUUAUAUGUGGCCCUUUCAAAAGCCCAAUUAUACAGAAAUCCGACAGUACUGCAAUCACUGGCGAAAUUUUGCUGACAUUGAUGAUUCCUGGAAAAGUAUAAAGAGUAUCUUGGACUGGACAUCUUUUAACCAGGAGAGAAUUGUUGAUGUUGCUGGACCAGGGGGUUGGAAUGACCCAGAUAUGUUAGUGAUUGGCAACUUUGGCCUCAGCUGGAAUCAGCAAGUAACUCAGAUGGCCCUCUGGGCUAUCAUGGCUGCUCCUUUAUUCAUGUCUAAUGACCUCCGACACAUCAGCCCUCAAGCCAAAGCUCUCCUUCAGGAUAAGGACGUAAUUGCCAUCAAUCAGGACCCCUUGGGCAAGCAAGGGUACCAGCUUAGACAGGGAGACAACUUUGAAGUGUGGGAACGACCUCUCUCAGGCUUAGCCUGGGCUGUAGCUAUGAUAAACCGGCAGGAGAUUGGUGGACCUCGCUCUUAUACCAUCGCAGUUGCUUCCCUGGGUAAAGGAGUGGCCUGUAAUCCUGCCUGCUUCAUCACACAGCUCCUCCCUGUGAAAAGGAAGCUAGGGUUCUAUGAAUGGACUUCAAGGUUAAGAAGUCACAUAAAUCCCACAGGCACUGUUUUGCUUCAGCUAGAAAAUACAAUGCAGAUGUCAUUAAAAGACUUACUUUAAY (SEQ ID NO: 2) X =GGGAUCCUACC (SEQ ID NO: 2) Y = UUUGAAUU (SEQ ID NO: 3)

Aliquots of 50 mg/mL ethanolic solutions of HGT5001, DOPE, cholesteroland DMG-PEG2K were mixed and diluted with ethanol to 3 mL final volume.Separately, an aqueous buffered solution (10 mM citrate/150 mM NaCl, pH4.5) of GLA mRNA was prepared from a 1 mg/mL stock. The lipid solutionwas injected rapidly into the aqueous mRNA solution and shaken to yielda final suspension in 20% ethanol. The resulting nanoparticle suspensionwas filtered, diafiltrated with 1×PBS (pH 7.4), concentrated and storedat 2-8° C. Final concentration=0.68 mg/mL GLA mRNA (encapsulated).Z_(ave)=79.6 nm (Dv₍₅₀₎=57.26 nm; Dv₍₉₀₎=100 nm).

Example 6

Lipid nanoparticles comprising HGT5001, DOPE, cholesterol andDMG-PEG2000 and encapsulating human erythropoietin (EPO) mRNA wereformed via standard ethanol injection methods. (Ponsa, et al., Int. J.Pharm. (1993) 95: 51-56.) Ethanolic stock solutions of the lipids wereprepared ahead of time at a concentration of 50 mg/mL and stored at −20°C.

Human erythropoietin (EPO) mRNA was synthesized by in vitrotranscription from a plasmid DNA template encoding the gene, which wasfollowed by the addition of a 5′ cap structure (Cap1) (Fechter, P. etal., J. Gen. Virology (2005) 86: 1239-1249) and a 3′ poly(A) tail ofapproximately 200 nucleotides in length as determined by gelelectrophoresis. The 5′ and 3′ untranslated regions present in the humanerythropoietin (EPO) mRNA are respectively represented as X and Y in SEQID NO: 1, as indicated below.

Human Erythropoietin mRNA: SEQ ID NO: 1XAUGGGGGUGCACGAAUGUCCUGCCUGGCUGUGGCUUCUCCUGUCCCUGCUGUCGCUCCCUCUGGGCCUCCCAGUCCUGGGCGCCCCACCACGCCUCAUCUGUGACAGCCGAGUCCUGGAGAGGUACCUCUUGGAGGCCAAGGAGGCCGAGAAUAUCACGACGGGCUGUGCUGAACACUGCAGCUUGAAUGAGAAUAUCACUGUCCCAGACACCAAAGUUAAUUUCUAUGCCUGGAAGAGGAUGGAGGUCGGGCAGCAGGCCGUAGAAGUCUGGCAGGGCCUGGCCCUGCUGUCGGAAGCUGUCCUGCGGGGCCAGGCCCUGUUGGUCAACUCUUCCCAGCCGUGGGAGCCCCUGCAGCUGCAUGUGGAUAAAGCCGUCAGUGGCCUUCGCAGCCUCACCACUCUGCUUCGGGCUCUGGGAGCCCAGAAGGAAGCCAUCUCCCCUCCAGAUGCGGCCUCAGCUGCUCCACUCCGAACAAUCACUGCUGACACUUUCCGCAAACUCUUCCGAGUCUACUCCAAUUUCCUCCGGGGAAAGCUGAAGCUGUACACAGGGGAGGCCUGCAGGACAGGGGACAGAUGAY X = GGGAUCCUACC (SEQ ID NO: 2) Y =UUUGAAUU (SEQ ID NO: 3)

Aliquots of 50 mg/mL ethanolic solutions of the HGT5001, DOPE,cholesterol and DMG-PEG2K were mixed and diluted with ethanol to 3 mLfinal volume. Separately, an aqueous buffered solution (10 mMcitrate/150 mM NaCl, pH 4.5) of EPO mRNA was prepared from the 1 mg/mLstock. The lipid solution was injected rapidly into the aqueous mRNAsolution and shaken to yield a final suspension in 20% ethanol. Theresulting nanoparticle suspension was filtered, diafiltrated with 1×PBS(pH 7.4), concentrated and stored at 2-8° C. Final concentration=1.09mg/mL EPO mRNA (encapsulated). Z_(ave)=62.1 nm (Dv₍₅₀₎=45.2 nm;Dv₍₉₀₎=74.6 nm).

Example 7

To determine whether the HGT5000-based lipid nanoparticles encapsulatinghuman GLA mRNA and prepared in accordance with Example 4 above werecapable of delivering encapsulated polynucleotide constructs to one ormore target cells, a dose response study was conducted in wild type(CD-1) mice that were subsequently monitored for human GLA proteinproduction.

The foregoing studies were performed using male or female CD-1 mice ofapproximately 6-8 weeks of age at the beginning of each experiment.Samples were introduced over a one week period at day 1 and again at day5 by a single bolus tail-vein injection. The serum concentrations of GLAprotein were determined at six hours, twenty-four hours, forty-eighthours and seventy-two hours following the administration of the secondintravenous dose. Mice were sacrificed seventy-two hours following theadministration of the second intravenous dose on day eight and organswere perfused with saline. The liver, spleen and when applicable, thebrain, of each mouse was harvested, apportioned into two parts andstored in either 10% neutral buffered formalin or snap-frozen and storedat 80° C.

As illustrated in FIG. 1, following the intravenous injection of two 10μg, 20 μg, 30 μg, 60 μg or 90 μg doses of GLA mRNA loaded in theHGT5000-based lipid nanoparticles, a substantial level of human GLAprotein could be detected in mouse serum within 6 hours. Furthermore,detectable levels of GLA protein could be observed in the serum 48 hoursfollowing intravenous administration of the second single dose. Asillustrated in FIG. 2, nanogram levels of human GLA protein were alsodetectable in select organs of the mice, such as the liver, kidney andspleen 72 hours following the administration of the second bolustail-vein injection of GLA mRNA.

Additional studies evaluating the HGT5000-based lipid nanoparticlesencapsulating human GLA mRNA and prepared in accordance with Example 4above were also performed using a murine model of Fabry disease. Sampleswere introduced by a single bolus 90 μg dose (based on encapsulated GLA)of the GLA-loaded lipid nanoparticle via a tail-vein injection.Supraphysiological levels of GLA protein (approximately 50 times higher)were detected in the serum 24 hours post-administration of the single 90ug dose of GLA. As illustrated in FIG. 3 and FIG. 4, human GLA proteinwas detectable in the serum and in select organs of the Fabry micefollowing the administration of a bolus tail-vein injection of theHGT5000-based lipid nanoparticle encapsulating GLA mRNA. In particular,human GLA protein was detected in the serum of the Fabry mice followingadministration of the GLA mRNA-loaded HGT5000-based lipid nanoparticlesover a 72 hour time period. Human GLA protein levels were alsodetectable in select Fabry mouse organs following the administration ofthe GLA mRNA-loaded HGT5000-based lipid nanoparticles both at 24 hoursand 72 hours post-administration.

Example 8

To determine whether the HGT5001-based lipid nanoparticles encapsulatinghuman GLA mRNA and prepared in accordance with Example 5 above werecapable of delivering encapsulated polynucleotide constructs to one ormore target cells, a dose response study was conducted in wild type(CD-1) mice that were subsequently monitored for human GLA proteinproduction.

The foregoing studies were performed using male or female CD-1 mice ofapproximately 6-8 weeks of age at the beginning of each experiment.Samples were introduced by a single bolus tail-vein injection. Mice weresacrificed at designated time points and organs were perfused withsaline. The liver, spleen and when applicable, the brain, of each mousewas harvested, apportioned into two parts and stored in either 10%neutral buffered formalin or snap-frozen and stored at −80° C.

The a single 30 μg dose of the HGT50001-based GLA mRNA-loaded lipidnanoparticles were administered to the wild type mice, and asillustrated in FIG. 5 at 6 hours post-administration, human GLA proteinwas detected in serum at concentrations that exceeded normalphysiological levels by 100-fold. As also depicted in FIG. 5,twenty-four hours following administration of the HGT5001-based GLAmRNA-loaded lipid nanoparticles to the wild type mice, human GLA proteinremained detectable at concentrations that exceeded normal physiologicalconcentrations by 30-fold higher. Further, as depicted in FIG. 6,substantial concentrations of human GLA protein could be detected in theliver, kidney and spleen of the wild-type mice after treatmenttwenty-four hours post administration of the HGT5001-based GLAmRNA-loaded lipid nanoparticles.

Example 9

The instant study was conducted to further demonstrate the ability ofboth the HGT5000-based and the HGT5001-based lipid nanoparticles todeliver encapsulated human erythropoietin (EPO) mRNA to one or moretarget cells in wild-type Sprague Dawley rats. HGT5000 andHGT50001-based EPO mRNA-loaded lipid nanoparticles were prepared inaccordance with the protocols set forth in the foregoing examples.Samples were administered by a single bolus tail-vein injection. Theconcentration of EPO protein secreted into the bloodstream was monitoredover a twenty-four hour time period, with serum samples obtained at six,twelve, eighteen and twenty-four hours following administration.

Human EPO protein was detected in the Sprague-Dawley rat serum followingadministration of the EPO mRNA-loaded HGT5000- and HGT5001-based lipidnanoparticles over a twenty-four hour time period. As shown in FIG. 7,both HGT5000-based and HGT5001-based lipid nanoparticles resulted inefficacious protein production in the wild-type Sprague Dawley rats.Significant levels of human EPO protein were detected over the course ofthis study for both HGT5000 and HGT5001-based nanoparticle systems.Accordingly, the present example illustrates that both HGT5000- andHGT5001-based lipid nanoparticles provide highly efficacious means ofdelivering polynucleotide constructs to one or more target cells andthat following expression of such lipid nanoparticles to such targetcells, the expressed protein encoded by the encapsulated mRNA wasdetectable in serum.

DISCUSSION

The foregoing studies illustrate that the lipid compounds disclosedherein are useful as liposomal delivery vehicles or as components ofliposomal delivery vehicles. In particular, such compounds andcompositions facilitate the delivery encapsulated polynucleotides (e.g.,mRNA polynucleotides encoding functional proteins or enzymes) to one ormore target cells, tissues and organs, thereby causing such cells toexpress the encapsulated polynucleotide. For example, following a singleintravenous injection of a given dose of an mRNA polynucleotideencapsulated in an HGT5000-based lipid nanoparticle, a substantialconcentration of the encoded protein was detected in both serum and inone or more target organs of the subject mice. Furthermore, as evidentby Example 8, in many instances the concentration of expressed proteinwell exceeded those concentrations necessary for therapeutic efficacy,therefore suggesting that only a fraction of the administered dose ofthe compositions are necessary to achieve therapeutically effectiveconcentrations within the plasma, target organ, tissue or cells. As aresult, the total administered amount of cationic lipid that isnecessary to deliver a therapeutically effective amount of theencapsulated agent may be reduced, resulting in a correspondingreduction in the toxicity of the compositions.

We claim:
 1. A lipid nanoparticle comprising: mRNA; and a cationic lipidhaving the following structure


2. The lipid nanoparticle of claim 1, further comprising one or morelipids selected from the group consisting of a cationic lipid, a neutrallipid, a PEG-modified lipid, a non-cationic lipid and a helper lipid. 3.The lipid nanoparticle of claim 1, further comprising C14-DMG-PEG2000,DOPE and cholesterol.
 4. A method of treating disease in a subject,wherein the method comprises administering an effective amount of thelipid nanoparticle of claim 1 to the subject.
 5. A method oftransfecting one or more target cells with a polynucleotide, wherein themethod comprises contacting the one or more target cells with the lipidnanoparticle of claim 1 such that the one or more target cells aretransfected with the polynucleotide.
 6. The lipid nanoparticle of claim1, wherein the mRNA encodes an enzyme or a protein.
 7. The lipidnanoparticle of claim 6, wherein the mRNA encodes the protein or enzymeselected from the group consisting of human growth hormone,erythropoietin, al-antitrypsin, acid alpha glucosidase, arylsulfatase A,carboxypeptidase N, α-galactosidase A, alpha-L-iduronidase,iduronate-2-sulfatase, iduronate sulfatase,N-acetylglucosamine-1-phosphate transferase, N-acetylglucosaminidase,alpha-glucosaminide acetyltransferase, N-acetylglucosamine 6-sulfatase,N-acetylgalactosamine-4-sulfatase, beta-glucosidase, galactose-6-sulfatesulfatase, beta-galactosidase, beta-glucuronidase, glucocerebrosidase,heparan sulfamidase, heparin-N-sulfatase, lysosomal acid lipase,hyaluronidase, galactocerebrosidase, ornithine transcarbamylase (OTC),carbamoyl-phosphate synthetase 1 (CPS1), argininosuccinate synthetase(ASS1), argininosuccinate lyase (ASL), arginase 1 (ARG1), cysticfibrosis transmembrane conductance regulator (CFTR), survival motorneuron (SMN), Factor VIII, Factor IX and low density lipoproteinreceptors (LDLR).
 8. The lipid nanoparticle of claim 1, furthercomprising one or more helper lipids, non-cationic lipids, andPEG-modified lipid components.
 9. The lipid nanoparticle of claim 1,further comprising DOPE, cholesterol, and/or DMG-PEG2000.
 10. A lipidnanoparticle comprising: mRNA; and a cationic lipid having thestructure,


11. The lipid nanoparticle of claim 10, further comprising one or morelipids selected from the group consisting of a cationic lipid, a neutrallipid, a PEG-modified lipid, a non-cationic lipid and a helper lipid.12. The lipid nanoparticle of claim 10, further comprisingC14-DMG-PEG2000, DOPE and cholesterol.
 13. A method of treating diseasein a subject, wherein the method comprises administering an effectiveamount of the lipid nanoparticle of claim 10 to the subject.
 14. Amethod of transfecting one or more target cells with a polynucleotide,wherein the method comprises contacting the one or more target cellswith the lipid nanoparticle of claim 10 such that the one or more targetcells are transfected with the polynucleotide.
 15. The lipidnanoparticle of claim 10, wherein the mRNA encodes an enzyme or aprotein.
 16. The lipid nanoparticle of claim 15, wherein the mRNAencodes the protein or enzyme selected from the group consisting ofhuman growth hormone, erythropoietin, al-antitrypsin, acid alphaglucosidase, arylsulfatase A, carboxypeptidase N, α-galactosidase A,alpha-L-iduronidase, iduronate-2-sulfatase, iduronate sulfatase,N-acetylglucosamine-1-phosphate transferase, N-acetylglucosaminidase,alpha-glucosaminide acetyltransferase, N-acetylglucosamine 6-sulfatase,N-acetylgalactosamine-4-sulfatase, beta-glucosidase, galactose-6-sulfatesulfatase, beta-galactosidase, beta-glucuronidase, glucocerebrosidase,heparan sulfamidase, heparin-N-sulfatase, lysosomal acid lipase,hyaluronidase, galactocerebrosidase, ornithine transcarbamylase (OTC),carbamoyl-phosphate synthetase 1 (CPS1), argininosuccinate synthetase(ASS1), argininosuccinate lyase (ASL), arginase 1 (ARG1), cysticfibrosis transmembrane conductance regulator (CFTR), survival motorneuron (SMN), Factor VIII, Factor IX and low density lipoproteinreceptors (LDLR).
 17. The lipid nanoparticle of claim 10, furthercomprising one or more helper lipids, non-cationic lipids, andPEG-modified lipid components.
 18. The lipid nanoparticle of claim 10,further comprising DOPE, cholesterol, and/or DMG-PEG2000.